• 沒有找到結果。

Expression of recombinant anticoagulant hirudin in the differentiated cultures of the porcine mammary epithelial cell line SI-PMEC

N/A
N/A
Protected

Academic year: 2021

Share "Expression of recombinant anticoagulant hirudin in the differentiated cultures of the porcine mammary epithelial cell line SI-PMEC"

Copied!
9
0
0

加載中.... (立即查看全文)

全文

(1)

Expression of recombinant anticoagulant hirudin in the differentiated

cultures of the porcine mammary epithelial cell line SI-PMEC

Y.L. Sun

a

, Y.C. Chou

a

, T.C. Kuan

b

, C.F. Tu

a

, C.S. Lin

b,

*

a

Division of Biotechnology, Animal Technology Institute, Taiwan, P.O. Box 23, Chunan 35099, Miaoli, Taiwan

bDepartment of Biological Science and Technology, National Chiao Tung University, No. 75 Po-Ai Street, Hsinchu 30068, Taiwan

Received 7 September 2007; revised 22 October 2007; accepted 25 February 2008

Abstract

To express recombinant proteins in the spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) currently established in our laboratory, a chemically synthesized DNA fragment encoding the anticoagulant hirudin was used to construct a mammalian expression vec-tor under the control of the goat b-casein regulavec-tory sequence. The vecvec-tor, named pGB562/Hi, was transfected into the SI-PMEC cells to yield pGB562/Hi/SI-PMEC. The pGB562/Hi/SI-PMEC cells expressed recombinant hirudin only when they were differentiated into functional struc-tures by growth on a Matrigel-coated petri dish supplemented with the lactogenic hormone prolactin. The differentiated pGB562/Hi/SI-PMEC cells produced about 0.5e0.6 mg of recombinant hirudin/mg of total cellular protein. These results suggest that the established SI-PMEC cells have pharmaceutical potential to inducibly express bioactive heterogeneous proteins.

Ó 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Recombinant protein; Mammary gland; Epithelial cell; SI-PMEC; Hirudin

1. Introduction

We have established a spontaneously immortalized porcine mammary epithelial cell line, SI-PMEC, from the mammary gland of a lactating sow and maintained it in culture long-term by continuous subculturing (Sun et al., 2005, 2006). SI-PMEC cells can differentiate into functional structures and will express and secrete milk proteins when cultured on Matri-gel-coated petri dishes supplemented with lactogenic hormone

(Sun et al., 2006). It is unclear, however, whether the

SI-PMEC cells can potentially express exogenous recombinant genes and produce bioactive proteins, as it had not been assessed prior to this study.

The anti-thrombotic polypeptide hirudin, which is isolated from the salivary glands of Hirudo medicinalis, contains 64e66 amino acids (Markwardt, 1970; Harvey et al., 1986). Hirudin binds specifically to thrombin, a protein involved in the blood clotting cascade, and thereby inhibits coagulation

(Dodt et al., 1984; Seemu¨ller et al., 1986). Therefore,

recombi-nant hirudin is useful for treating diseases related to the coag-ulation activity of thrombin or for preventing, alleviating or ameliorating symptoms of these diseases, which include acute coronary syndromes (Matheson and Goa, 2000; Weitz and

Bates, 2003; Greinacher, 2004).

In the present study, we sought to determine whether a syn-thetic hirudin gene could be transfected into the porcine mam-mary epithelial cell line, SI-PMEC, and subsequently induced to generate bioactive recombinant hirudin. We also character-ized the inducing factors and expression potential of the bio-active heterogeneous protein in the SI-PMEC culture system.

Abbreviations: SI-PMEC, spontaneously immortalized porcine mammary epithelial cell line; RT-PCR, reverse-transcription polymerase chain reaction; GB562, a DNA fragment (5.62 kb) containing the 50flanking sequence and

in-tron 1 of the goat b-casein gene; Hi, Hirudin; EGFP, enhanced green fluores-cent protein, a reporter used in gene transfection; pGB562/GFP, an expression vector containing an EGFP encoding gene fragment which is controlled under the sequence of GB562; pGB562/Hi, an expression vector containing a hirudin encoding gene fragment which is controlled under the sequence of GB562; pGB562/GFP/SI-PMEC, the SI-PMEC cells transfected with the expression vector of pGB562/GFP; pGB562/Hi/SI-PMEC, the SI-PMEC cells transfected with the expression vector of pGB562/Hi.

* Corresponding author. Tel.:þ886 3 5131338; fax: þ886 3 5729288. E-mail address:lincs@mail.nctu.edu.tw(C.S. Lin).

1065-6995/$ - see front matterÓ 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2008.02.004

(2)

2. Materials and methods 2.1. Cell culture

The culture of SI-PMEC cells was performed as in our pre-viously reported protocol (Sun et al., 2006). For cell growth and proliferation, the cells were cultured with the basal me-dium, DMEM/F12 (GIBCO, Carlsbad, CA, USA) containing 10% fetal calf serum (batch number #716543; Biological In-dustries, Kibbutz Beit Haemek, IL, USA), insulin (10 mg/ml; Sigma, Canton, MA, USA), and hydrocortisone (1 mg/ml; Sigma), in a humidified atmosphere containing 5% CO2 at 37C. The fetal calf serum was pretreated by heat inactivation at 56C for 30 min before use. For cell differentiation with secretory function, the cells were cultured onto petri dishes coated with BD MatrigelÔ (BD Biosciences Clontech, Frank-lin Lakes, NJ, USA) and the basal medium was supplied with prolactin (5 mg/ml; Sigma).

2.2. Synthesis of the DNA fragment encoding hirudin

The DNA fragment encoding hirudin was synthesized according to the method reported by Kochanowski et al.

(2006) with slight modifications. Based on the sequence of

the hirudin gene disclosed in GenBank accession number M12693, two oligonucleotides, Hi-1 (123-mer) and Hi-2 (128-mer) were designed to build the gene encoding hirudin, wherein 15-mer at the 30-end of both strands are complemen-tary to each other. In addition, two primers, Hi-PCR-F (33-mer) and Hi-PCR-R (32-(33-mer), were designed according to the 50-terminal sequences of the above Hi-1 and Hi-2 oligonu-cleotides and enabled to clone the gene by restriction endonu-cleases, SalI and NotI (Table 1 and Fig. 1A). All of the oligonucleotides were synthesized by Applied Biosystems (Foster City, CA, USA).

A reaction mixture containing 8 ml of each the oligonucle-otides Hi-1 and Hi-2 (10 mM of each), 10 ml dNTPs (2 mM), 0.5 U Taq DNA polymerase (HT Biotechnology, Cambridge, UK), 5 ml of 10 PCR buffer (15 mM MgCl2, 500 mM KCl, 1% Triton X-100, 0.1% gelatin and 100 mM TriseHCl, pH 7.9), and 18 ml H2O, was incubated at 70C for 30 min. The primers Hi-PCR-F and Hi-PCR-R were used to amplify the gene encoding hirudin as well. The reaction mixture was

heated at 94C for 5 min before entering the PCR cycles in an Applied Biosystems DNA thermal cycler (Applied Biosys-tems). The reaction conditions were 94C for 45 s, 60C for 45 s, and 72C for 45 s. After a total of 36 cycles, the mixture was then subjected to 72C for 10 min to complete the DNA extension.

2.3. Construction of the hirudin expression vector

The construction flowchart of the hirudin expression vector is shown in Fig. 1A. The vector of pGB562/GFP (10.40 kb), a mammary gland-specific expression vector which contains 5.62 kb of the 50flanking sequence and intron 1 of goat b-casein gene linked to a gene fragment encoding enhanced green fluo-rescent protein (EGFP) (Wu et al., 2003), was used in this study as a control of DNA transfection and expression as well as also providing the regulatory region of goat b-casein gene to tran-scribe EGFP or hirudin mRNA. The pGB562/GFP vector was treated with SalI and NotI to remove the EGFP DNA fragment and then was ligated with the synthesized hirudin gene fragment which was also digested with SalI and NotI to obtain a hirudin expression vector, pGB562/Hi, which can specifically express hirudin in differentiated mammary epithelial cells.

2.4. Gene transfection of SI-PMEC

The SI-PMEC cells were transfected with pEGFP-N1 (a vec-tor constructed with a CMV promoter followed by the EGFP gene) (BD Biosciences Clontech), pGB562/Hi or pGB562/ GFP by electroporation. After mixing the plasmid DNA (2e 4 mg) with SI-PEMC cells (1 106cells in 0.4 ml), the mixture was put into an electroporation cuvette 0.4 cm in width and treated by an electroporator (BTX 830; Gentronics, San Diego, CA, USA) under the following conditions: 200e500 mV/cm at 50 mV intervals, 99 ms, and pulsed 4 times. The treated cells were moved into a 60 mm petri dish and cultured with basal me-dium at 37C in a 5% CO2atmosphere for 4 h, and then replaced with fresh medium. After 24 h culturing, the expression of GFP in the DNA transfected SI-PMEC cells was observed using a fluorescence microscope (Axiovert 135; Carl Zeiss, Gottingen, Germany) with fluorescein isothiocyanate (FITC) optics.

Geneticin (G418; 500 mg/ml; Sigma) was also used for se-lection of transfected SI-PMEC cells, and the culture medium

Table 1

Sequences of oligonucleotides used to synthesis of full-length DNA fragment encoding hirudin

Oligonucleotide Sequence (50/30) Number of mer

Template oligonucleotides

Hi-1 gatccttt atg gtt gtt tac act gac tgc act gaa tcc ggt cag aac ctg tgc ctg tgc gaa ggc tct aac gtt tgc ggc cag ggc aac aaa tgc atc ctg ggc tct gat ggc gaa aaa aat c

123 Hi-2 ggccgcccta tta ttg cag gta ttc ttc cgg gat ttc ttc aaa gtc gcc gtc gtt gtg aga ctg cgg ttt cgg

agt acc ttc gcc agt aac gca ttg att ttt ttc gcc atc aga gcc cag gat gca t

128

Primer oligonucleotides

Hi-PCR-F tcggtcgacttt atg gtt gtt tac act gac tgc (SalI) 33 Hi-PCR-R catgcggccgcccta tta ttg cag gta ttc tt (NotI) 32

The start codon ATG designed in the synthesized hirudin gene is indicated with bold letters in the Hi-1 and Hi-2 sequences. The arranged cutting sites by restriction enzyme SalI and NotI are indicated with bold letters in the Hi-PCR-F and Hi-PCR-R sequences, respectively.

(3)

was replaced irregularly thereafter. After one successive gen-eration, SI-PMEC cells that stably contained pGB562/Hi or pGB562/GFP (pGB562/Hi/SI-PMEC or pGB562/GFP/SI-PMEC) were obtained.

2.5. Determination of b-casein and hirudin transcripts For determining b-casein, a major milk protein, and hirudin gene expression induced by cultured substrata and lactogenic hormone, the pGB562/Hi/SI-PMEC cells cultured on a Matri-gel-coated petri dish in basal medium containing pro-lactin (5 mg/ml; Sigma) were used to prepare total cellular RNA for reverse transcription-polymerase chain reaction

(RT-PCR). The PCR primers for amplification of b-casein and hirudin transcripts were as follows:

(1) Porcine b-casein (GenBank accession no. X54974)

b-ca-F (21-mer) 50-CCA AAG CTA AGG AGA CCA

TTG-30

b-ca-R (19-mer) 50-CAA CTG GTT GAG GCA CAG G-30 (2) Hirudin (GenBank accession no. M12693)

Hi-F (21-mer) 50-TAC ACT GAC TGC ACT GAA TCC -30 Hi-R (18-mer) 50-TTG CAG GTA TTC TTC CGG-30 Total cellular RNA preparation and RT-PCR were per-formed as previously described (Lin and Hsu, 2005). Total

Digested with Sal I and Not I Recovered the larger DNA fragment of vector DNA ligation

Digested with Sal I and Not I Recovered the DNA fragment Transformation Sal I Not I pGB562/GFP 10.40 kb Sal I Not I pGB562/Hi 9.92 kb Sal I Not I

DNA fragment coding hirudin (198 bp)

Not I Hi-1 Hi-2 Hi-PCR-F Hi-PCR-R Sal I (228 bp) PCR

5’-tcggtcgacttt ATG GTT GTT TAC ACT GAC TGC ACT GAA TCC GGT CAG Met Val Val Tyr Thr Asp Cys Thr G1u Ser G1y G1n AAC CTG TGC CTG TGC GAA GGC TCT AAC GTT TGC GGC CAG GGC AAC AAA Asn Leu Cys Leu Cys G1u G1y Ser Asn Val Cys G1y G1n G1y Asn Lys TGC ATC CTG GGC TCT AGA GGC GAA AAA AAT CAA TGC GTT ACT GGC GAA Cys 11e Leu G1y Ser Asp G1y G1u Lys Asn G1n Cys Val Thr G1y G1u GGT ACT CCC AAA CCG CAG TCT CAC AAC GAC GGC GAC TTT GAA GAA ATC G1y Thr Pro Lys Pro G1n Ser His Asn Asp G1y Asp Phe G1u G1u 11e CCG GAA GAA TAC CTG CAA TAA tagggcggccgcatg-3’

Pro G1u G1u Tyr Leu G1n Stop

5’-tcggtcgacttt ATG GTT GTT TAC ACT GAC TGC ACT GAA TCC GGT CAG Met Val Val Tyr Thr Asp Cys Thr G1u Ser G1y G1n AAC CTG TGC CTG TGC GAA GGC TCT AAC GTT TGC GGC CAG GGC AAC AAA Asn Leu Cys Leu Cys G1u G1y Ser Asn Val Cys G1y G1n G1y Asn Lys TGC ATC CTG GGC TCT AGA GGC GAA AAA AAT CAA TGC GTT ACT GGC GAA Cys 11e Leu G1y Ser Asp G1y G1u Lys Asn G1n Cys Val Thr G1y G1u GGT ACT CCC AAA CCG CAG TCT CAC AAC GAC GGC GAC TTT GAA GAA ATC G1y Thr Pro Lys Pro G1n Ser His Asn Asp G1y Asp Phe G1u G1u 11e CCG GAA GAA TAC CTG CAA TAA tagggcggccgcatg-3’

Pro G1u G1u Tyr Leu G1n Stop

5’-tcggtcgacttt ATG GTT GTT TAC ACT GAC TGC ACT GAA TCC GGT CAG Met Val Val Tyr Thr Asp Cys Thr G1u Ser G1y G1n AAC CTG TGC CTG TGC GAA GGC TCT AAC GTT TGC GGC CAG GGC AAC AAA Asn Leu Cys Leu Cys G1u G1y Ser Asn Val Cys G1y G1n G1y Asn Lys TGC ATC CTG GGC TCT AGA GGC GAA AAA AAT CAA TGC GTT ACT GGC GAA Cys 11e Leu G1y Ser Asp G1y G1u Lys Asn G1n Cys Val Thr G1y G1u GGT ACT CCC AAA CCG CAG TCT CAC AAC GAC GGC GAC TTT GAA GAA ATC G1y Thr Pro Lys Pro G1n Ser His Asn Asp G1y Asp Phe G1u G1u 11e CCG GAA GAA TAC CTG CAA TAA tagggcggccgcatg-3’

Pro G1u G1u Tyr Leu G1n Stop Sal I

Not I

A

B

Fig. 1. Schematic presentation of the procedure to synthesize the DNA fragment encoding hirudin and to construct the expression vector, pGB562/Hi. (A) The synthesized Hi-1 and Hi-2 were used as templates and the primer pair, Hi-PCR-F and Hi-PCR-R, was used to amplify the DNA fragment (228 bp) by PCR. Restriction enzyme SalI and NotI sites were designed at the 50 and 30 termini in the DNA fragment, respectively. The amplified 228 bp fragment contained a 198 bp hirudin gene. The construction and features of the vector are detailed in the Section2. Abbreviations: pGB562, a DNA fragment containing the 50flanking sequence and intron 1 of goat b-casein gene; Ap, ampicillin resistance gene; Kan/Neo, kanamycin and neomycin resistance gene; Hi, hirudin variant I coding gene; Ori, replication origin from pUC plasmid. (B) The nucleotide sequences of the synthesized DNA fragment encoding hirudin. The nucleotide sequences listed are under the predicted coding arrangement. The amino acid sequence deduced from the DNA sequence was shown under the corresponding codons.

(4)

RNA was extracted from SI-PMEC (1 106cells) using Tri-zol (GIBCO) in accordance with the manufacturer’s instruc-tions. Five micrograms of the DNase I (Promega, Madison, WI, USA) treated total RNA were reverse-transcribed using 2.5 mM oligo-dT primers (Promega), 1 mM of each dNTP (Promega), 20 U ribonuclease inhibitor (HT Biotechnology) and 5 U reverse transcriptase (HT Biotechnology) in RT buffer (25 mM TriseHCl, pH 8.3, 50 mM KCl, 2 mM DTT and 5 mM MgCl2) in a total volume of 20 ml at 39C for 60 min. For each PCR reaction, 3 ml of RT product were added to a final volume of 50 ml containing 10 mM TriseHCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl, 0.01% Triton X-100, 0.001% gelatin, 200 mM of each dNTP, 0.5 U Taq DNA poly-merase (HT Biotechnology) and 0.2 mM of each primer pair. For the gene expression assay, PCR was carried out for 28 cycles with 30 s denaturation at 94C, 45 s annealing at 55C, and 45 s extension at 72C.

2.6. Western blot assay

Cultured SI-PMEC cells were recovered to prepare homo-geneous extract by sonication and centrifugation. The total amount of protein in homogeneous extract was measured by the Bradford dye binding assay (BioRad Laboratories, Hercu-les, CA, USA) and bovine serum albumin as the standard.

The Western blot was performed as in our previous report

(Pan et al., 2007). Equal amounts of protein and the

commer-cial hirudin standard (Recombinant hirudin #377853, vial of 200 mg, ca. 2000 ATU; Calbiochem, EMD Biosciences, Darm-stadt, Germany) were separated by SDS-polyacrylamide gel electrophoresis (NuPAGE 4e12% Bis-Tris Gel, Invitrogen, Carlsbad, CA, USA). After electrophoresis, the proteins were transferred to Nitropure membrane (Micron Separations, Westborough, MA, USA), and then blotted with the mouse monoclonal hirudin antibody (diluted 1:2000; Abcam, Cam-bridge, UK) for 2 h. After washing with 1 D-PBS, the mem-brane was incubated with secondary antibody of anti-mouse IgG conjugated with alkaline phosphatase (AP) (diluted 1:2500; Sigma), and then developed with AP Immunoblot Assay Developer (BioRad Laboratories).

2.7. Immunocytochemistry

The SI-PMEC cells were plated in 6-well plates (1 105 cells/well) for 24 h and then fixed in 95% ethanol for 1 min. Twenty microliters of mouse monoclonal hirudin antibody (di-luted 1:500) were applied per well and the plates were incu-bated at 37C for 1 h in a 100% humidity chamber. The plates were then washed (3 5 min) with D-PBS at room temperature and the cells were incubated with secondary antibody using a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s rec-ommendations. The incubation and washes were as described above. Finally, the cells were stained with a Vector TMB Sub-strate Kit (Vector Laboratories) and examined by light micros-copy (Sun et al., 2006).

2.8. Assay of anti-coagulation activity of hirudin

The hirudin activity in the homogeneous extract was deter-mined through anti-thrombin activity using a chromogenic substrate, Chromozyme TH (tosyl-Gly-Pro-Arg-p-nitroanilide; Roche, Mannheim, Germany) (Sohn et al., 1991; Kim et al., 2001). The amidolytic cleavage of Chromozyme TH by thrombin (Sigma) was measured as the rate of increase in ab-sorbance at 405 nm with a microtiter plate reader (Bio-Tek, Winooski, VT, USA). Thrombin (0.6 NIH unit/ml), diluted culture supernatant and Chromozyme TH (200 mM) were loaded into the 96-well assay plate simultaneously. The reac-tion was monitored every 30 s for 5 min. One anti-thrombin activity (ATU) was defined as the amount of hirudin able to completely inhibit one NIH unit of human thrombin at 37C. Commercial recombinant hirudin (Calbiochem) was used as a standard to estimate the hirudin concentration by the activity measurement (Sohn et al., 1991).

3. Results

3.1. Construction of the hirudin expression vector

Synthesis of the gene encoding hirudin was based on two designed oligonucleotides, Hi-1 and Hi-2, for which the codon sequences corresponded to the amino acid sequences found in natural hirudins, with a change to add an initiating ATG codon at the 50-terminal of the open reading frame of hirudin

(Fig. 1A). The size of the DNA fragment amplified by PCR

using the Hi-PCR-F and Hi-PCR-R as primer pair and the hybrid DNA of Hi-1 and Hi-2 as template was 228 bp. The restriction enzyme-treated (SalI and NotI) synthesized DNA fragment (including an open reading frame covering 198 bp) was inserted into a vector with a 50 flanking sequence and intron 1 of the goat b-casein gene as a regulatory sequence (5.62 kb) to yield the construct pGB562/Hi (9.92 kb)

(Fig. 1A). The inserted hirudin gene was sequenced and was

identical to the original design as shown inFig. 1B.

3.2. Gene transfection and GFP expression in SI-PMEC cells

Plasmid pEGFP-N1 was used to transfect SI-PMEC cells to test the efficiency of gene transfection via electroporation and the effect on cell survival. Cell survival was generally de-creased in the range from 95 to 24%, and the efficiency of transfection generally increased from 1.2 to 17.1% when the electroporation voltage was increased from 200 to 500 mV at 50 mV intervals. These efficiencies were lower than that of pEGFP-N1 transfected into primary epithelial cells of the por-cine mammary gland (Sun et al., 2005). However, the popula-tion of EGFP-expressing SI-PMEC cells could optimally reach over 30% after one passage with G418 selection. This trans-fection efficiency was acceptable for further experiments involving pGB562/GFP and pGB562/Hi transfection and for determining the optimal conditions for inducible expression

(5)

of GFP and hirudin in the pGB562/GFP/SI-PMEC and pGB562/Hi/SI-PMEC cells, respectively.

3.3. Inducible GFP expression in pGB562/GFP/SI-PMEC cells

The SI-PMEC cells transfected with the mammary gland-specific expression vector pGB562/GFP (named pGB562/ GFP/SI-PMEC cells) were used to test the potential to express recombinant protein in transfected SI-PMEC cells. The pGB562/GFP/SI-PMEC cells showed the characteristic cob-blestone morphology of epithelial cells (Fig. 2A); the fluores-cent signal of the culture could not be observed under the fluorescence microscope with FITC optics (Fig. 2B) when the cells were routinely cultured in basal medium on a petri dish. However, when the pGB562/GFP/SI-PMEC cells were cultured on a Matrigel-coated petri dish and the basal medium was supplemented with prolactin, the cells differentiated and formed a functional morphology; that is, the cells aggregated to form alveolar- and duct-like structures (Fig. 2C). With continued incubation of the cultures, the duct-like structures were not only maintained but continued to develop into a tubular network with lumens (data not shown). These differen-tiated pGB562/GFP/SI-PMEC cells produced strong GFP fluorescence (Fig. 2D). These results indicate that recombinant protein was inducible and could be produced in SI-PMEC after transfecting with a mammary gland-specific expression vector.

3.4. Expression of recombinant hirudin in pGB562/Hi/ SI-PMEC cells

Hirudin expression in pGB562/Hi/SI-PMEC cells was detected by immunocytochemistry, Western blotting and RT-PCR. Immunocytochemistry showed very little hirudin ex-pression when the cells were cultured in basal medium on petri dishes (Fig. 2E); however, hirudin was strongly expressed when pGB562/Hi/SI-PMEC cells were cultured on Matrigel-coated dishes supplemented with prolactin (Fig. 2F). The ex-pression of recombinant hirudin in pGB562/Hi/SI-PMEC cells was confirmed by Western blotting (Fig. 3). Hirudin was detected in the differentiated pGB562/Hi/SI-PMEC cells and exhibited the same molecular mass in the electrophoretic SDS-polyacrylamide gel (18e20 kDa) as the commercial re-combinant hirudin.

Furthermore, hirudin expression at the transcription level was detected by RT-PCR (Fig. 4). The transcription results are basically consistent with the translation results. In addition to morphology, expression of milk protein transcripts is also a significant marker to determine the status of SI-PMEC cell differentiation. Therefore, b-casein and hirudin transcripts were evaluated in this study. When the pGB562/Hi/SI-PMEC cells were grown on a plastic substratum, the b-casein transcript was detected at a very low level and hirudin was un-detectable. In contrast, when the cells were cultured on a Ma-trigel-coated petri dish supplemented with 5 mg/ml prolactin, the b-casein and hirudin were strongly expressed (Fig. 4).

3.5. Bioactivity assay of hirudin expressed by pGB562/Hi/SI-PMEC cells

The anti-coagulation bioactivity of hirudin in homogenate of pGB562/Hi/SI-PMEC cells was assessed. The bioactivity of expressed recombinant hirudin was evaluated by determin-ing absorption changes at 405 nm caused by inhibition of the thrombin reaction using Chromozyme TH. Commercial hiru-din was used to establish a standard curve (from 0 to 60 ng/ ml at 15 ng/ml intervals) (Fig. 5A). Based on the standard curve and the measured values, hirudin in the cells was quan-tified. As shown in Fig. 5B, activity equivalent to the back-ground was detected in the homogenate prepared from undifferentiated pGB562/Hi/SI-PMEC cells. The results of ar-tificially adding hirudin into the cell homogenate indicate that the detected hirudin activity would not be affected by the hirudin in the homogenate. These results indicate that this method could be used to determine hirudin production in SI-PMEC cell homogenate. The production of recombinant hirudin in the differentiated pGB562/Hi/SI-PMEC cells was es-timated to be 0.5e0.6 mg/mg of total cellular protein (Fig. 5B). The activity of hirudin in the milk stored at 4C in a refriger-ator for 1 week was decreased by approximate 25% compared with that of the fresh milk sample containing recombinant hiru-din. We proposed that the decreased activity of hirudin in the stored milk might due to degradation of the hirudin or conjuga-tion with milk proteins during storage.

3.6. Effect of Matrigel and prolactin on recombinant protein expression

The effects of Matrigel and prolactin on recombinant hirudin expression in pGB562/Hi/SI-PMEC cells were determined. The pGB562/Hi/SI-PMEC cells were cultured in the basal medium without or with supplemental prolactin (5 mg/ml) and grown on petri dishes lacking or containing Matrigel coating. Re-combinant hirudin production in the pGB562/Hi/SI-PMEC cultures was enhanced about 12- and 21-fold by prolactin and Matrigel, respectively (Fig. 5C). The recombinant hirudin pro-duction was enhanced 29-fold when the pGB562/Hi/SI-PMEC cells were cultured on a Matrigel-coated petri dish and supple-mented with prolactin.

4. Discussion

This study describes the expression of a heterogeneous pro-tein, hirudin, in the immortalized porcine mammary epithelial cell line, SI-PMEC. The hirudin gene was synthesized and used to construct a mammary expression vector controlled under the regulatory sequences of goat b-casein, a tissue-and stage-specific expression promoter (Wu et al., 2003;

Rijn-kels and Pieper, 1989). Thus, hirudin was expressed upon

differentiation of hirudin gene-transfected SI-PMCE cells into functional structures induced by growth on a Matrigel-coated dish supplemented with prolactin.

b-Casein is the most abundant protein in the milk of goats and cows. Accordingly, it has been suggested that the b-casein

(6)

gene is a powerful promoter that may be suitable for use in driving transgene expression in the mammary glands of trans-genic animals (Roberts et al., 1992; Ebert et al., 1994). Several elements, including composite response element (CoRE), sig-nal transducers and activators of transcription (STAT), CCAAT/enhancer binding protein (C/EBPs), glucocorticoid response element (GRE), pregnancy-specific mammary nu-clear factor (PMF) and octamer-binding protein (Oct), within the promoter region of the gene were identified by motif assay or experimental study (Groner and Gouilleux, 1995;

Gorodet-sky and Bremel, 1998; Rosen et al., 1999). In addition to being

in the regulatory region of the gene, intron 1 is also essential for the gene regulation. Kang et al. (1998) indicated that

prolactin-inducible enhancer activity was localized in intron 1 of the bovine b-casein gene. In addition, several elements in intron 1 of the bovine b-casein gene interact cooperatively not only with each other, but also with its promoter for hor-monal induction (Petitclerc et al., 1995). The present results suggest that the 6.2-kb regulative sequence of the goat b-casein gene is sufficient and effective for directing exogenous gene expression in the SI-PMECs. The results were confirmed by the report fromWu et al. (2003).

A number of immortal mammary epithelial cell lines of cow (German and Barash, 2002; Rose et al., 2002) have been established. The cell lines could be used to produce bio-active recombinant protein. Lazaris et al. (2002) used the

Fig. 2. Inducible targeting expression in SI-PMEC cells transfected with pGB562/GFP (pGB562/GFP/SI-PMEC) and pGB562/Hi (pGB562/Hi/SI-PMEC). (A) An image under bright field of pGB562/GFP/SI-PMEC cells cultured in basal medium. (B) The image under a fluorescent field, which is the same field shown in panel (A). (C) An image under a bright field of pGB562/GFP/SI-PMEC cells cultured on a Matrigel-coated petri dish and basal medium provided with prolactin. Mor-phological differentiation (duct-like structures developed into a tubular network with lumens) of pGB562/GFP/SI-PMEC cells cultured with Matrigel and supple-mental prolactin was observed. (D) The image under the fluorescent field is the same field shown in panel (C). (E) The undifferentiated pGB562/Hi/SI-PMEC cells cultured in basal medium and treated with a mouse monoclonal antibody against hirudin. The cells produced no immunopositive signal. (F) pGB562/Hi/SI-PMEC cells cultured on a Matrigel-coated petri dish and basal medium supplemented with prolactin and treated with anti-hirudin monoclonal antibody. The many cells that formed duct-like structures developed into a tubular network with lumens and were immunopositive (deep brown color) for hirudin, indicating that the recombinant hirudin was expressed in these functional-structural pGB562/Hi/SI-PMEC cells. Bars indicate 200 mm.

(7)

immortalized bovine mammary epithelial MAC-T cells to pro-duce soluble recombinant spider silk.Neumann et al. (2006) also used a similar system to produce recombinant equine pro-relaxin in transfected MAC-T cells and this propro-relaxin could induce a biological response in anin vitro bioassay.

Recombinant hirudins have been produced using a variety of expression systems, including yeast (Saccharomyces cerevi-siae and Pichia pastoris) (Sohn et al., 1991; Kim et al., 2001;

Zhou and Zhang, 2002; Shi et al., 2006), bacteria (Escherichia

coli) (Kochanowski et al., 2006; Tan et al., 2002), and in mam-malian cell lines (CHO and HBK) (Riesbeck et al., 1998;

Guarna et al., 2000). In the present study, recombinant hirudin

was expressed in an immortalized porcine mammary epithelial cell line, SI-PMEC. The SI-PMEC cells have all the machin-ery to properly fold a mammalian protein, which is important for biological activity.

Of interest in this study was that pGB562/Hi/SI-PMEC cells, which transfected with a mammary gland-specific ex-pression vector pGB562/Hi (Fig. 1A), produced recombinant protein with a molecular mass of 18e20 kDa as determined by Western blotting. In fact, a similar phenomenon was also shown for the commercial recombinant hirudin (Rade et al., 1999). The immunoreactive bands present in the commercial hirudin and the homogenate of SI-PMEC cells did not agree with the predicted 6e7 kDa form of hirudin. It can be pro-posed that the band was indeed present, with a molecular mass corresponding to that of hirudin trimers identified by the monoclonal anti-hirudin antibody. This speculation is con-sistent with the report byRade et al. (1999).

Optimal expression of recombinant hirudin by SI-PMEC cells requires not only stromal Matrigel, which supports differ-entiation by organizing the basement membrane and providing growth factors, but also stimulation by lactogenic hormones. Our results indicate that the most important factor for enhanc-ing production of recombinant protein in SI-PMEC is Matrigel rather than prolactin. Wu et al. (2003)reported that prolactin plays a major role in inducing milk protein gene expression in the mouse mammary epithelial cell line NMuMG. In our previous study, we showed that prolactin is essential and can strongly increase milk gene expression in primary porcine mammary epithelial cells (Sun et al., 2005). Contrary to the previous results, in the present study we demonstrated that the established porcine mammary epithelial cell line, SI-PMEC, can be triggered to differentiate, form functional structures and express milk genes without prolactin supple-mentation. However, providing a suitable substratum is neces-sary for triggering differentiation of SI-PMEC cells. The genetic modifications in the SI-PMEC need to be further ex-plored before the differential regulation of the cell is conclu-sively understood.

In this study, we focused on producing bioactive recombi-nant hirudin in SI-PMEC cells. Additional studies are needed to determine optimal conditions for secretion of the nant proteins in the SI-PMEC system. The yields of recombi-nant proteins produced by the immortalized bovine mammary epithelial MAC-T cells are variable. The yield of recombinant equine prorelaxin in MAC-T cells was only 4 ng/ml (Neumann

et al., 2006). However, the recombinant spider silk produced

by MAC-T cells could reach 20e50 ng/ml (Lazaris et al., 2002). In addition to using the SI-PMEC system to produce recombinant proteins, successful in vitro expression of re-combinant product in a porcine mammary epithelial cell line also can be used for an in vitro screening system to identify superior transgenes prior to transfer, thereby improving the ef-ficiency of transgenic livestock generation to produce pharma-ceutically relevant peptides in milk. By targeting expression to

1 2 3

B

M 2

A

22 KDa 17 KDa 3 1

Fig. 3. Western blot analysis of recombinant hirudin expressed in pGB562/Hi/ SI-PMEC cells. (A) The samples were subjected to 12% SDS-polyacrylamide gel electrophoresis under reducing conditions. (B) The parallel samples in (A) were separated on an SDS-polyacrylamide gel, transferred to a nylon mem-brane and then detected by enhanced chemiluminescence using a mouse monoclonal antibody against hirudin. M, a protein marker; molecular masses at 17 and 22 kDa are indicated; lane 1, the homogenate (20 mg) from pGB562/ Hi/SI-PMEC cells cultured in basal medium and grown on a petri dish; lane 2, the homogenate (10 mg) from pGB562/Hi/SI-PMEC cells cultured on a Matri-gel-coated petri dish and basal medium supplemented with 5 mg/ml prolactin; lane 3, 5 ng of commercial recombinant hirudin. In lanes 2 and 3, bands immunopositive for hirudin were detected. Hirudin is indicated by arrows.

M 2 3 4 5 6 7 8 400 200 (bp) 300 Hirudin (198 bp) 1 ββ-casein (341 bp)

Fig. 4. Hirudin and b-casein gene expression in pGB562/Hi/SI-PMEC cells. The SI-PMEC cells were cultured in basal medium without supplemental pro-lactin on a petri dish (lanes 1 and 2) and in basal medium with supplemental prolactin on a Matrigel-coated petri dish (lanes 3 and 4). The pGB562/Hi/SI-PMEC cells were cultured in basal medium without supplemental prolactin on a petri dish (lanes 5 and 6) and in basal medium with supplemental prolactin on a Matrigel-coated petri dish (lanes 7 and 8). The amplified b-casein (341 bp) and hirudin (198 bp) DNA fragments are indicated. M, a 100-bp ladder and the molecular masses are indicated.

(8)

the mammary gland, several heterologous recombinant human proteins have been produced from the milk of transgenic live-stock, including that of goats, sheep, cattle and rabbits (

Hou-debine, 2000; Niemann and Kues, 2003). The biological

activity of the recombinant proteins such as human anti-throm-bin III, a-antitrypsin and tissue plasminogen activator were as-sessed and the therapeutic effects have been characterized

(Meade et al., 1999; Rudolph, 1999). The proteins from the

milk of transgenic livestock are expected to be on the market within the next few years (Niemann and Kues, 2003).

In summary, our study reports the potential of gene trans-fection by electroporation in our established porcine mammary epithelial cell line, SI-PMEC. The hirudin transgene was suc-cessfully expressed in differentiated SI-PMEC cells, which

formed functional structures. Anti-coagulation bioactivity was detected for the produced recombinant hirudin. Thus, we have provided experimental data to demonstrate that SI-PMEC cells have potential to produce pharmaceutically relevant peptides. Acknowledgements

The authors gratefully acknowledge Prof. MC Huang at the National Chung Hsing University and Dr ST Wu at the Na-tional Chiayi University in Taiwan for the gift of pGB562/ GFP plasmid. This work was supported by the grant of 92-EC-17-A-17-R7-0454 from the Ministry of Economic Affairs of Executive Yuan and the grant of NSC 96-2628-B-009-001-MY3 from National Science Council, Taiwan.

Δ Δ OD 405 /min Δ Δ OD 405 /min Hirudin (ng/ml) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.05 0.10 0.15 0.20 0 10 20 30 40 50 60 70 0 20 40 60 80 100 120 0.0 0.2 0.4 0.6 0.8 1 2 3 4 12 X 21 X 29 X Prolactin + + Matrigel + + Homogenate (μg of protein/ml) Hirudin ( g/mg protein of homogenate)

A

B

C

Fig. 5. Anti-coagulation activity assay of recombinant hirudin in the homogenate of pGB562/Hi/SI-PMEC cells and the effects of Matrigel and prolactin on the production of recombinant hirudin in the cells. (A) Thrombin titration curve of average residual thrombin activity plotted against increasing concentrations of the commercial recombinant hirudin from 0 to 60 ng/ml at 15 ng/ml intervals. The equation of the linear relationship between DOD405(y) and hirudin mass (x) is

y¼ 0.0041x, R2

¼ 0.9955. (B) The plots of anti-coagulation activity of the homogenates isolated from the undifferentiated pGB562/Hi/SI-PMEC cells (closed circles), the homogenates isolated from the undifferentiated pGB562/Hi/SI-PMEC cells which were artificially added with 30 ng commercial recombinant hirudin to each concentration of the detected samples (open circles), and the homogenates isolated from the differentiated pGB562/Hi/SI-PMEC cells that were cultured in basal medium with supplemental prolactin (5 m/ml) and grown on a Matrigel-coated dish (closed squares). (C) The anti-coagulation activity of the homogenates isolated from each treatment was determined and the hirudin content was calculated. Treatment 1, the homogenate isolated from pGB562/Hi/SI-PMEC cells cul-tured in basal medium and grown on a plastic dish; treatment 2, from pGB562/Hi/SI-PMEC cells culcul-tured in basal medium with supplemental prolactin (5 mg/ml) and grown on a plastic dish; treatment 3, from pGB562/Hi/SI-PMEC cells cultured in basal medium and grown on a Matrigel-coated dish; treatment 4, from pGB562/Hi/SI-PMEC cells cultured in basal medium with supplemental prolactin and grown on a Matrigel-coated dish. Each value indicates the mean SE of three independent determinations.

(9)

References

Dodt J, Mu¨ller HP, Seemu¨ller U, Chang JY. The complete amino acid sequence of hirudin, a thrombin specific inhibitor: application of colour carboxyme-thylation. FEBS Lett 1984;165:180e4.

Ebert KM, DiTullio P, Barry CA, Schindler JE, Ayres SL, Smith TE, et al. Induction of human tissue plasminogen activator in the mammary gland of transgenic goats. Biotechnology (NY) 1994;12:699e702.

German T, Barash I. Characterization of an epithelial cell line from bovine mammary gland. In Vitro Cell Dev Biol Anim 2002;38:282e92. Gorodetsky SI, Bremel R. The factors of tissue-specific expression of the

bo-vine b-casein gene. In: Castro FO, Janne J, editors. Mammary gland trans-genesis: therapeutic protein production. New York: Springer-Verlag; 1998. Greinacher A. Lepirudin: a bivalent direct thrombin inhibitor for

anticoagula-tion therapy. Expert Rev Cardiovasc Ther 2004;2:339e57.

Groner B, Gouilleux F. Prolactin-mediated gene activation in mammary epi-thelial cells. Curr Opin Genet Dev 1995;5:587e94. doi:10.1016/0959-437X(95)80027-1.

Guarna MM, Cote HC, Kwan EM, Rintoul GL, Meyhack B, Heim J, et al. Fac-tor X fusion proteins: improved production and use in the releasein vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein. Protein Expr Purif 2000;20:133e41.

Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP, Courtney M, et al. Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis. Proc Natl Acad Sci U S A 1986;83:1084e8.

Houdebine LM. Transgenic animal bioreactors. Transgenic Res 2000;9: 305e20.

Kang YK, Lee CS, Chung AS, Lee KK. Prolactin-inducible enhancer activity of the first intron of the bovine b-casein gene. Mol Cell 1998;8:259e65. Kim MD, Yoo YJ, Seo JH. Enhanced transformation efficiency of an an-ticoagulant hirudin gene intoSaccharomyces cerevisiae by a double d-sequence. J Microbiol Biotechnol 2001;11:61e4.

Kochanowski R, Kotlowski R, Szweda P. Novel method of expression and purification of hirudin based on pBAD TOPO, pTYB12 vectors and gene synthesis. Protein Expr Purif 2006;50:25e30. doi:10.1016/ j.pep.2006.06.004.

Lazaris A, Arcidiacono S, Huang Y, Zhou JF, Duguay F, Chretien N, et al. Spi-der silk fibers spun from soluble recombinant silk produced in mammalian cells. Science 2002;295:472e6.

Lin CS, Hsu CW. Differentially transcribed genes in skeletal muscle of duroc and taoyuan pigs. J Anim Sci 2005;83:2075e86.

Markwardt F. Hirudin as an inhibitor of thrombin. Methods Enzymol 1970;19: 924e32.

Matheson AJ, Goa KL. Desirudin: a review of its use in the management of thrombotic disorders. Drugs 2000;60:679e700.

Meade HM, Echelard Y, Ziomek CA, Young MW, Harvey M, Cole ES, et al. Expression of recombinant proteins in the milk of transgenic animals. In: Fernandez JM, Hoeffler JP, editors. Gene expression systems. San Diego, CA: Academic Press; 1999. p. 399e427.

Neumann JL, Lazaris A, Huang YJ, Karatzas C, Ryan PL, Bagnell CA. Produc-tion and characterizaProduc-tion of recombinant equine prorelaxin. Domest Anim Endocrinol 2006;31:173e85. doi:10.1016/j.domaniend.2005.10.001. Niemann H, Kues WA. Application of transgenesis in livestock for agriculture

and biomedicine. Anim Reprod Sci 2003;79:291e317. doi:10.1016/ S0378-4320(03)00169-6.

Pan CH, Lin JL, Lai LP, Chen CL, Huang SKS, Lin CS. Downregulation of angiotensin converting enzyme II is associated with pacing-induced sus-tained atrial fibrillation. FEBS Lett 2007;581:526e34.

Petitclerc D, Attal J, The´ron MC, Bearzotti M, Bolifraud P, Kann G, et al. The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice. J Biotechnol 1995;40:169e78. doi:10.1016/0168-1656(95)00047-T.

Rade JJ, Cheung M, Miyamoto S, Dichek DA. Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins. Gene Ther 1999;6:385e92.

Riesbeck K, Chen D, Kemball-Cook G, McVey JH, George AJ, Tuddenham EG, et al. Expression of hirudin fusion proteins in mammalian cells: a strategy for prevention of intravascular thrombosis. Circulation 1998;98:2744e52.

Rijnkels M, Pieper FR. Casein gene-based mammary gland-specific transgene expression. In: Castro FO, Janne J, editors. Mammary gland transgenesis: therapeutic protein production. New York: Springer-Verlag; 1989. p. 41e64. Roberts B, DiTullio P, Vitale J, Hehir K, Gordon K. Cloning of the goat beta-casein-encoding gene and expression in transgenic mice. Gene 1992;121:255e62. Rose MT, Aso H, Yonekura S, Komatsu T, Hagino A, Ozutsumi K, et al.In

vitro differentiation of a cloned bovine mammary epithelial cell. J Dairy Res 2002;69:345e55.

Rosen JM, Wyszomierski SL, Hadsell D. Regulation of milk protein gene ex-pression. Annu Rev Nutr 1999;19:407e36.

Rudolph NS. Biopharmaceutical production in transgenic livestock. Trends Bi-otechnol 1999;17:367e74. doi:10.1016/S0167-7799(99)01341-4. Seemu¨ller U, Dodt J, Fink E, Fritz H. Proteinase inhibitors of the leech Hirudo

medicinalis (hirudins, bdellins, eglins). In: Barrett AJ, Salvesen G, editors. Proteases. Amsterdam: Elsevier; 1986. p. 337e59.

Shi B, Li J, Yu A, Yuan B, Wu B. Two-step ion-exchange chromatographic purification of recombinant hirudin-II and its C-terminal-truncated deriva-tives expressed in Pichia pastoris. Process Biochem 2006;41:2446e51. doi:10.1016/j.procbio.2006.07.018.

Sohn JH, Lee SK, Choi ES, Rhee SK. Gene expression and secretion of the anticoagulant hirudin inSaccharomyces cerevisiae. J Microbiol Biotechnol 1991;1:266e73.

Sun YL, Lin CS, Chou YC. Establishment and characterization of a spontane-ously immortalized porcine mammary epithelial cell line. Cell Biol Int 2006;30:970e6. doi:10.1016/j.cellbi.2006.06.023.

Sun YL, Lin CS, Chou YC. Gene transfection and expression in a primary cul-ture of mammary epithelial cells isolated from lactating sows. Cell Biol Int 2005;29:576e82. doi:10.1016/j.cellbi.2005.03.021.

Tan S, Wu W, Liu J, Kong Y, Pu Y, Yuan R. Efficient expression and secretion of recombinant hirudin III inE. coli using theL-asparaginase II signal se-quence. Protein Expr Purif 2002;25:430e6.

Weitz JI, Bates ER. Direct thrombin inhibitors in cardiac disease. Cardiovasc Toxicol 2003;3:13e25.

Wu HT, Lin CS, Huang MC.In vitro and ex vivo green fluorescent protein ex-pression in alveolar mammary epithelial cells and mammary glands driven by the distal 50-regulative sequence and intron 1 of the goat beta-casein gene. Reprod Fertil Dev 2003;15:231e9.

Zhou XS, Zhang YX. Decrease of proteolytic degradation of recombinant hir-udin produced byPichia pastoris by controlling the specific growth rate. Biotechnol Lett 2002;24:1449e53.

數據

Fig. 1. Schematic presentation of the procedure to synthesize the DNA fragment encoding hirudin and to construct the expression vector, pGB562/Hi
Fig. 2. Inducible targeting expression in SI-PMEC cells transfected with pGB562/GFP (pGB562/GFP/SI-PMEC) and pGB562/Hi (pGB562/Hi/SI-PMEC)
Fig. 3. Western blot analysis of recombinant hirudin expressed in pGB562/Hi/ SI-PMEC cells
Fig. 5. Anti-coagulation activity assay of recombinant hirudin in the homogenate of pGB562/Hi/SI-PMEC cells and the effects of Matrigel and prolactin on the production of recombinant hirudin in the cells

參考文獻

相關文件

Animal or vegetable fats and oils and their fractiors, boiled, oxidised, dehydrated, sulphurised, blown, polymerised by heat in vacuum or in inert gas or otherwise chemically

Milk and cream, in powder, granule or other solid form, of a fat content, by weight, exceeding 1.5%, not containing added sugar or other sweetening matter.

For 5 to be the precise limit of f(x) as x approaches 3, we must not only be able to bring the difference between f(x) and 5 below each of these three numbers; we must be able

[This function is named after the electrical engineer Oliver Heaviside (1850–1925) and can be used to describe an electric current that is switched on at time t = 0.] Its graph

The resulting color at a spot reveals the relative levels of expression of a particular gene in the two samples, which may be from different tissues or the same tissue under

6 《中論·觀因緣品》,《佛藏要籍選刊》第 9 冊,上海古籍出版社 1994 年版,第 1

The first row shows the eyespot with white inner ring, black middle ring, and yellow outer ring in Bicyclus anynana.. The second row provides the eyespot with black inner ring

In Case 1, we first deflate the zero eigenvalues to infinity and then apply the JD method to the deflated system to locate a small group of positive eigenvalues (15-20