中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/25116

行政院國家科學委員會專題研究計畫 期中進度報告

粒線體融合素/GTP 脢在非小細胞肺癌病人中過度表現的預

後意義(1/3)

計畫類別： 個別型計畫

計畫編號：

NSC91-2314-B-039-018-執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日

執行單位： 中國醫藥學院醫學系

計畫主持人： 許南榮

計畫參與人員： 周寬基 姜淑芬 關智尤 林景屏

報告類型： 精簡報告

處理方式： 本計畫可公開查詢

中

華

民

國 92 年 5 月 14 日

行政院國家科學委員會補助專題研究計畫þ期中

進度報告

※※※※※※※※※※※※※※※※※※※※※※※※※※※※※

※※※※※

※

※

※

※

※ 粒線體融合素/GTP 在 非 小 細 胞 肺 癌 病 人中

過

度

表

現

的

預

後

意義 ※

※

※

※

※

※ Prognostic significance of mitofusin/GTPase overexpression

in ※

※ patients with non-small cell lung cancer

※

※

※

※

※

※※※※※※※※※※※※※※※※※※※※※※※※※※※※※

※※※※※

計畫類別：

þ

個別型計畫

□整合型計畫

計畫編號：NSC91－2314－B－039－018－

執行期間：

91 年 08 月 01 日至

94 年 07 月 31 日

計畫主持人： 許 南 榮

計畫參與人員：周寬基 (

[email protected]

)

姜淑芬

關智尤

林景屏

成果報告類型：

þ

精簡報告

執行單位：中 國 醫 藥 學 院 附 設 醫 院 胸 腔 外 科 與 醫 學 研 究 部

研究計畫上所列預期完成之工作項目：

第一年，我們所要完成之工作項目 (91 年 08 月 01 日至 92 年 07 月 31 日)：

(1) To determine the expression of the respective mitofusin/GTPases by

RT-PCR;

(2) To subclone the respective mitofusin/GTPase gene into an expression vector,

to deliver recombinant DNA into bacterial cells to express recombinant

mitofusin/GTPases.

進度結果

1.

Comparison of amino acid sequences between GTPase and mitofusins

(1) GTPase (HSU95822);

(2) Mitofusin1 (AF329637)

;

(3) FLJ20693 fis (AK000700)

;

(4) Mitofusin2 (NM_014874)

(1) (2) (3) ---(4) --- --- --- --- --- ---MSLLF SRCNSIVTVK (1)

---(2) ----MAEPVS PLKHFVLAKK GITAIFDQLL EFVTEGSHFV EATYKNPELD RIATEDDLVE MQGYKDKLSI

(3) ----MAEPVS PLKHFVLAKK AITAVFDQLL EFVTEGSHFV EATYKNPELD RIATEDDLVE MQGYKDKLSI

(4) KNKRHMAEVN ASPLKHFVTA KKKINGIFEQ LGAYIQESAT FLEDTYRNAE LDPVTTEEQV LDVKGYLSKV

(1)

---(2) IGEVLSRRHM KVAFFGRTSS GKSSVINAML WDKVLPSGIG HITNCFLSVE GTDGDKAYLM TEGSDEKKSV

(3) IGEVLSRRHM KVAFFGRTSS GKSSVINAML WDKVLPSGIG HITNCFLSVE GTDGDKAYLM TEGSDEKKSV

(4) RGISEVLARR HMKVAFFGRT SNGKSTVINA MLWDKVLPSG IGHTTNCFLR VEGTDGHEAF LLTEGSEEKR

(1)

---(2) KTVNQLAHAL HMDKDLKAGC LVRVFWPKAK CALLRDDLVL VDSPGTDVTT ELDSWIDKFC LDADVFVLVA

(3) KTVNQLAHAL HMDKDLKAGC LVRVFWPKAK CALLRDDLVL VDSPGTDVTT ELDSWIDKFC LDADVFVLVA

(4) SAKTVNQLAH ALHQDKQLHA GSLVSVMWPN SKCPLLKDDL VLMDSPGIDV TTELDSWIDK FCLDADVFVL

(1) --- --- --- --- --TVNQLAHA LHMDKDLKAG CLVRVFCPKA

(2) NSESTLMNTE KHFFHKVNEW LSKPNIFILN NRWDASASEP EYMEDVRRQH MERCLHFLVE ELKVANALEA

(3) NSESTLMNTE KHFFHKVNER LSKPNIFILN NRWDASASEP EYMEDVRRQH MERCLHFLVE ELKVVNALEA

(4) VANSESTLMQ TEKHFFHKVS ERLSRPNIFI LNNRWDASAS EPEYMEEVRR QHMERCTSFL VDELGVVDRS

(1) KCALLRDDLV LVDSPGTDVT TELDSWIDKF CLDADVFVLV ANSESTLMNT EKHFFHKVNE RLSKPNIFIL

(2) QNRIFFVSAK EVLSARKQKA QGMPESGVAL AEGFHARLQE FQNFEQIFEE CISQSAVKTK FEQHTIRAKQ

(3) QNRIFFVSAK EVLSARKQKA QGMPESGVAL AEGFHARLQE FQNFEQIFEE CISQSAVKTK FEQHTIRAKQ

(4) QAGDRIFFVS AKEVLNARIQ KAQGMPEGGG ALAEGFQVRM FEFQNFERRF EECISQSAVK TKFEQHTVRA

(1) NNRWDASASE PEYMEDVRRQ HMERCLHFLV EELKVVNALE AQNRIFFVSA KEVLSARKQK AQGMPESGVA

(2) ILATVKNIMD SVNLAAEDKR HYSVEEREDQ IDRLDFIRNQ MNLLTLDVKK KIKEVTEEVA NKVSCAMTDE

(3) ILATVKNIMD SVNLAAEDKR FHVQ

(4) KQIAEAVRLI MDSLHMAARE QQVYCEEMRE ERQDRLKFID KQLELLAQDY KLRIKQITEE VERQVSTAMA

(1) LAEGFHARLQ EFQNFEQIFE ECISQSAVKT KFEQHTIRAK QILATVKNIM DSVNLAAEDK RHYSVEERED

(2) ICRLSVLVDE FCSEFHPNPD VLKIYKSELN KHIEDGMGRN LADRCTDEVN ALVLQTQQEI IENLKPLLPA

(4) EEIRRLSVLV DDYQMDFHPS PVVLKVYKNE LHRHIEEGLG RNMSDRCSTA ITNSLQTMQQ DMIDGLKPLL

(1) QIDRLDFIRN QMNLLTLDVK KKIKEVTEEV PNKVSCAMTD EICRLSVLVD EFCSEFHPNP DVLKIYKSLP

(2) GIQDKLHTLI PCKKFDLSYN LNYHKLCSDF QEDIVFRFSL GWSSLVHRFL GPRNAQRVLL GLSEPIFQLP

(4) PVSVRSQIDM LVPRQCFSLN YDLNCDKLCA DFQEDIEFHF SLGWTMLVNR FLGPKNSRRA LMGYNDQVQR

(1) RSLASTPTAP TTPATPDNAS QEELMITLVT GLASVTSRTS MGIIIVGGVI WKTIGWKLLS VSLTMYGALY

(2) RSLASTPTAP TTPATPDNAS QEELMITLVT GLASVTSRTS MGIIIVGGVI WKTIGWKLLS VSLTMYGALY

(4) PIPLTPANPS MPPLPQGSLT QEEFMVSMVT GLASLTSRTS MGILVVGGVV WKAVGWRLIA LSFGLYGLLY

putative transmembrane seq

(1) LYERLSWTTH AKERAFKQQF VNYATEKLRM IVSSTSANCS HQVKQQIATT FARLCQQVDI THKQLEEEIA

(2) LYERLSWTTH AKERAFKQQF VNYATEKLRM IVSSTSANCS HQVKQQIATT FARLCQQVDI TQKQLEEEIA

(4) VYERLTWTTK AKERAFKRQF VEHASEKLQL VISYTGSNCS HQVQQELSGT FAHLCQQVDV TRENLEQEIA

(1) RLPKEIDQLE KIQNNSKLLR NKAVQLENEL ENFTKQFLPS SNEES (2) RLPKEIDQLE KIQNNSKLLR NKAVQLENEL ENFTKQFLPS SNEES (4) AMNKKIEVLD SLQSKAKLLR NKAGWLDSEL NMFTHQYLQP SR

2.

DETERMINATION OF PRIMER SEQUENCES FOR MITOFUSIN1

5MFN1M:

5’-ATGGCAGAACCTGTTTCTCCACTGAATCAC-3’ (nts

1-26)

3MFN1M:

5’-CCTTTTATCTTCAGCTGCCAGGTTTACT-3’ (nts

1098-1070)

5MFN1:

5’-CTCCCTAGATCTTTAGCTTCTACTCCCA-3’ (nts

1664-1690)

3MFN1:

5’-TTAGGATTCTTCATTGCTTGAAGGTA-3’(nts

2226-2201)

Mf1M: 5MFN1M to 3MFN1M: 1098 – 0 = 1098 bp

Mf1S: 5MFN1 to 3MFN1: 2226 – 1663 = 563 bp

Mf1L: 5MFN1M to 3MFN1: 2226 – 0 = 2226 bp

3.

DETERMINATION OF PRIMER SEQUENCES FOR MITOFUSIN2

5MFN2a:

5’-ATGTCCCTGCTCTTCTCTCGATGCAA-3’ (nts 323-348)

3MFN2a:

5’-TGGCCACCAGCACAAACACATCAGC-3’ (nts1004-980)

5MFN2b:

5’-CTGTGACAAGCTGTGTGCTGACTTCC-3’ (nts 1909-1934)

3MFN2b:

5’-CTATGTGCTGGGCTGCAGGTACTGGTGTGTGA-3’(nts

2596-2565)

5MFN2a to 3MFN2a: 1004 – 322 = 682 bp

5MFN2b to 3MFN2b: 2596 – 1908 = 688 bp

5MFN2a to 3MFN2b: 2596 – 322 = 2274 bp

4.

Expression of mitofusins detected in lung cancer cells: lung cancer cells only

expressed Mfn1, but not Mfn2. The full-length Mfn1 cDNA contained an

endogenous Eco RI site.

H1437 H1648

Mf1M Mf1S Mf1M Mf1S Mf1L/EcoRI

M1

sEpH X2

M2

5.

Confirmed RT-PCR results by sequence determination

(1) Full length MF1 (Mf1L) expressed in lung cancer cells

Clone name: MF1-SP6, DNA sequence determined by ABI

TCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTG

GAATTC

GCCCTTATGGCA

GAACCTGTTTCTCCACTGAATCACTTTGTGCTGGCTAAGAAGGCGATTACTGCAATCTTTGACCAGTTA

CTGGAGTTTGTTACTGAAGGATCACATTTTGTTGAAGCAACATATAAGAATCCGGAACTTGATCGAATA

563 bp

1676 bp

1098 bp

852 bp

GCCACTGAAGATGATCTGGTAGAAATGCAAGGATATAAAGACAAGCTTTCCATCATTGGTGAGGTGCTA

TCTCGGAGACACATGAAGGTGGCATTTTTTGGCAGGACAAGCAGTGGGAAGAGCTCTGTTATCAATGCA

ATGTTGTGGGATAAAGTTCTCCCTAGTGGGATTGGCCATATAACCAATTGCTTCCTAAGCGTTGAAGGA

ACTGATGGAGATAAAGCCTATCTTATGACAGAAGGATCAGATGAAAAAAAGAGTGTGAAGACAGTTAAT

CAACTGGCCCATGCCCTTCACATGGACAAAGATTTGAAAGCTGGCTGTCTTGTACGTGTGTTTTGGCCA

AAAGCAAAATGTGCCCTCTTGAGAGATGACCTGGTGTTAGTAGACAGTCCAGGCACAGATGTCACTACA

GAGCTGGATAGCTGGATTGATAAGTTCTGCCTAGATGCTGATGTCTTTGTTTTGGGTCGCAAAACTCTG

AAATCANCACCTATTGAATACCGGGAAAACCCCTTTTTTTCACAAGGGTGAGTGACCGGCTTTCCCAGC

CCTAAATTTTCATTCCCAATATTCTGTGGGAGTCCCTGCATGAGCCCGAATTTGAAACGTCCGACGCAT

ACGAGCACAATCTGGGGAACTAGGTGAAGTGAGAACAACTTCTNNGTTCACGGANTNGTAAAAAGGATA

AGTGCCGAGCCCANATACTAAGTCGAGCACGTGCATCCTCCTGTCTCCGCGACATATCGCTGCATATAC

CCCGTAAA

NCBI blast ID:

1017650931-6968-32156

>gi|12744895|gb|AF329637.1|AF329637 Homo sapiens mitofusin 1 mRNA,

nuclear gene for mitochondrial protein, complete cds Length = 2226; Identities =

601/613 (98%)

Query: 6 atggcagaacctgtttctccactgaatcactttgtgctggctaagaaggcgattactgca 65 |||||||||||||||||||||||||| |||||||||||||||||||||| |||||||||| Sbjct: 1 atggcagaacctgtttctccactgaagcactttgtgctggctaagaaggggattactgca 60 Query: 66 atctttgaccagttactggagtttgttactgaaggatcacattttgttgaagcaacatat 125 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 61 atctttgaccagttactggagtttgttactgaaggatcacattttgttgaagcaacatat 120 Query: 126 aagaatccggaacttgatcgaatagccactgaagatgatctggtagaaatgcaaggatat 185 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 121 aagaatccggaacttgatcgaatagccactgaagatgatct ggtagaaatgcaaggatat 180 Query: 186 aaagacaagctttccatcattggtgaggtgctatctcggagacacatgaaggtggcattt 245 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 181 aaagacaagctttccatcattggtgaggtgctatctcggagacacatgaaggtggcattt 240 Query: 246 tttggcaggacaagcagtgggaagagctctgttatcaatgcaatgttgtgggataaagtt 305 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 241 tttggcaggacaagcagtgggaagagctctgttatcaatgcaatgttgtgggataaagtt 300 Query: 306 ctccctagtgggattggccatataac caattgcttcctaagcgttgaaggaactgatgga 365 ||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||| Sbjct: 301 ctccctagtgggattggccatataaccaattgcttcctaagtgttgaaggaactgatgga 360 Query: 366 gataaagcctatcttatgacagaaggatcagatgnnnnnnngagtgtgaagacagttaat 425 |||||||||||||||||||||||||||||||||| ||||||||||||||||||| Sbjct: 361 gataaagcctatcttatgacagaaggatcagatgaaaaaaagagtgtgaagacagttaat 420 Query: 426 caactggcccatgcccttcacatggacaaagatttgaaagctggctgtcttgtacgtgtg 485 |||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| Sbjct: 421 caactggcccatgcccttcacatggacaaagatttgaaggctggctgtcttgtacgtgtg 480

Query: 486 ttttggccaaaagcaaaatgtgccctcttgagagatgacctggtgttagtagacagtcca 545 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 481 ttttggccaaaagcaaaatgtgccctcttgagagatgacctggtgttagtagacagtcca 540 Query: 546 ggcacagatgtcactacagagctggatagctggattgataagttctgcctagatgctgat 605 |||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||| Sbjct: 541 ggcacagatgtcactacagagctggatagctggattgataagttttgcctagatgctgat 600 Query: 606 gtctttgttttgg 618 ||||||||||||| Sbjct: 601 gtctttgttttgg 613

Clone name: MF1-T7

TAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCA

GAATTC

GCCCTTTTAGGATTCTTCAT

TGCTTGAAGGTAGAAACTGCTTAGTAAAATTCTCCAGCTCATTTTCAAGTTGAACAGCTTTATTTCTTA

AGAGCTTTGAATTGTTTTGTATTTTCTCCAACTGATCTATTTCTTTGGGTAATCTAGCAATTTCTTCTT

CCAGCTGTTTTTGAGTAATATCAACTTGTTGGCACAGGCGAGCAAAAGTGGTAGCTATTTGTTGTTTTA

CTTGGTGACTGCAGTTTGCACTCGTGGAGCTAACAATCATCCTCAGTTTTTCAGTTGCATAGTTTACAA

ACTGCTGTTTAAAGGCTCGCTCCTTGGCATGGGTGGTCCAGCTCAGTCTTTCATAAAGATACAAGGCTC

CATACATAGTTAATGAAACAGATAGGAGTTTCCAGCCTATAGTTTTCCAAATCACTCCTCCAACAATAA

TGATGCCCATAGAAGTTCTAGATGTAACGGACGCCAATCCTGTTACTAATGTAATCATGAGTTCTTCCT

GTGATGCATTATCTGGCGTTGCTGGAGTGGTAGGAGCAGTGGGGTAGAAGCTAAAGATCTAGGGAGACC

TTTATATATTTTTAATACATCTGGGATTAGGATGAAACTCTGAACAAAATCATCACCCAAACAGACAGT

CGACAATTTCATCTGTCATTGCCATGAACTTGGTTGCACCTCTCGTACTCCTGAATTTTCTTACATCAG

GTAAGGTACTGGTCGATAGTAGTNCANTGCACCGAAGGCACGAATGCCTATTCCCGCGCAGGC

NCBI blast ID:

1017651107-8454-781

>gi|17447224|ref|XM_003183.6| Homo sapiens mitofusin 1 (MFN1), mRNA Length

= 2221

Identities = 562/564 (99%), Gaps = 1/564 (0%)

Query: 6 ttaggattcttcattgcttgaaggtagaaactgcttagtaaaattctccagctcattttc 65 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1255 ttaggattcttcattgcttgaaggtagaaactgcttagtaaaattctccagctcattttc 1196 Query: 66 aagttgaacagctttatttcttaagagctttgaattgttttgtattttctccaactgatc 125 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1195 aagttgaacagctttatttcttaagagctttgaattgttttgtattttctccaactgatc 1136 Query: 126 tatttctttgggtaatctagcaatttcttcttccagctgtttttgagtaatatcaacttg 185 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1135 tatttctttgggtaatctagcaatttcttcttccagctgtttttgagtaatatcaacttg 1076

Query: 186 ttggcacaggcgagcaaaagtggtagctatttgttgttttacttggtgactgcagtttgc 245 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1075 ttggcacaggcgagcaaaagtggtagctatttgttgttttacttggtgactgcagtttgc 1016 Query: 246 actcgtggagctaacaatcatcctcagtttttcagttgcatagtttacaaactgctgttt 305 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1015 actcgtggagctaacaatcatcctcagtttttcagttgcatagtttacaaactgctgttt 956 Query: 306 aaaggctcgctccttggcatgggtggtccagctcagtctttcataaagatacaaggctcc 365 |||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||| Sbjct: 955 aaaggctcgctccttggcatgggtggtccagctcagtctttcataaagatacaaagctcc 896 Query: 366 atacatagttaatgaaacagataggagtttccagcctatagttttccaaatcactcctcc 425 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 895 atacatagttaatgaaacagataggagtttccagcctatagttttccaaatcactcctcc 836 Query: 426 aacaataatgatgcccatagaagttctagatgtaacggacgccaatcctgttactaatgt 485 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 835 aacaataatgatgcccatagaagttctagatgtaacggacgccaatcctgttactaatgt 776 Query: 486 aatcatgagttcttcctgtgatgcattatctggcgttgctggagtggtaggagcagtggg 545 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 775 aatcatgagttcttcctgtgatgcattatctggcgttgctggagtggtaggagcagtggg 716 Query: 546 -gtagaagctaaagatctagggag 568 ||||||||||||||||||||||| Sbjct: 715 agtagaagctaaagatctagggag 692

(2) Full length MF2 (Mf2L) only expressed in esophageal cancer cells

Clone name: 81A M13F

CGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCG

GCCGCCAGTGTGATGGATATCTGCA

GAATTC

GGCTTATGTCCCTGCTCTTCTCTCGATGCAACTCTATC

GTCACAGTCAAGAAAAATAAGAGACACATGGCTGAGGTGAATGCATCCCCACTTAAGCACTTTGTCACT

GCCAAGAAGAAGATCAATGGCATTTTTGAGCAGCTGGGGGCCTACATCCAGGAGAGCGCCACCTTCCTT

GAAGACACGTACAGGAATGCAGAACTGGACCCCGTTACCACAGAAGAACAGGTTCTGGACGTCAAAGGT

TACCTATCCAAAGTGAGAGGCATCAGTGAGGTGCTGGCTCGGAGGCACATGAAAGTGGCTTTTTTTGGC

CGGACGAGCAATGGGAAGAGCACCGTGATCAATGCCATGCTCTGGGACAAAGTTCTGCCCTCTGGGATT

GGCCACACCACCAATTGCTTCCTGCGGGTAGAGGGCACAGATGGCCATGAGGCCTTTCTCCTTACCGAG

GGCTCAGAGGAAAAGAGGAGTGCCAAGACTGTGAACCAGCTGGCCCATGCCCTCCACCAGGACAAGCAG

CTCCATGCCGGCAGCCTAGTGAGTGTGATGTGGCCCAACTCTAAGTGCCCACTTCTGAAGGATGACCTC

GTTTTGATGGACAGCCCTGGTATTGATGTCACCACAGAGCTGGACAGCTGGATTGACAAGTTTTGTCTG

GATGCTGATGTGTTTGTGCTGGNGGCAAAGCCCNANTNCAA

NCBI blast ID: 1019091191-016662-20362

>gi|7662003|ref|NM_014874.1| Homo sapiens mitofusin 2 (MFN2), mRNA

Length = 4550

Identities = 672/680 (98%)

Query: 6 atgtccctgctcttctctcgatgcaactctatcgtcacagtcaagaaaaataagagacac 65 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 323 atgtccctgctcttctctcgatgcaactctatcgtcacagtcaagaaaaataagagacac 382 Query: 66 atggctgaggtgaatgcatccccacttaagcactttgtcactgccaagaagaagatcaat 125 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 383 atggctgaggtgaatgcatccccacttaagcactttgtcactgccaagaagaagatcaat 442 Query: 126 ggcatttttgagcagctgggggcctacatccaggagagcgccaccttccttgaagacacg 185 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 443 ggcatttttgagcagctgggggcctacatccaggagagcgccaccttccttgaagacacg 502 Query: 186 tacaggaatgcagaactggaccccgttaccacagaagaacaggttctggacgtcaaaggt 245 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 503 tacaggaatgcagaactggaccccgttaccacagaagaacaggttctggacgtcaaaggt 562 Query: 246 tacctatccaaagtgagaggcatcagtgaggtgctggctcggaggcacatgaaagtggcn 305 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 563 tacctatccaaagtgagaggcatcagtgaggtgctggctcggaggcacatgaaagtggct 622 Query: 306 nnnnnnggccggacgagcaatgggaagagcaccgtgatcaatgccatgctctgggacaaa 365 |||||||||||||||||||||| |||||||||||||||||||||||||||||||| Sbjct: 623 ttttttggccggacgagcaatgggaagagcaccgtgatcaatgccatgctctgggacaaa 682 Query: 366 gttctgccctctgggattggccacaccaccaattgcttcctgcgggtagagggcacagat 425 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 683 gttctgccctctgggattggccacaccaccaattgcttcctgcgggtagagggcacagat 742 Query: 426 ggccatgaggcctttctccttaccgagggctcagaggaaaagaggagtgccaagactgtg 485 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 743 ggccatgaggcctttctccttaccgagggctcagaggaaaagaggagtgccaagactgtg 802 Query: 486 aaccagctggcccatgccctccaccaggacaagcagctccatgccggcagcctagtgagt 545 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 803 aaccagctggcccatgccctccaccaggacaagcagctccatgccggcagcctagtgagt 862 Query: 546 gtgatgtggcccaactctaagtgcccacttctgaaggatgacctcgttttgatggacagc 605

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 863 gtgatgtggcccaactctaagtgcccacttctgaaggatgacctcgttttgatggacagc 922 Query: 606 cctggtattgatgtcaccacagagctggacagctggattgacaagttttgtctggatgct 665 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 923 cctggtattgatgtcaccacagagctggacagctggattgacaagttttgtctggatgct 982 Query: 666 gatgtgtttgtgctggnggc 685 |||||||||||||||| ||| Sbjct: 983 gatgtgtttgtgctggtggc 1002

Clone name: Kbb M13R

TTTAGGTGACACTATATAATACTCAAGCTATGCATCAAGCTTGGTACCGAGCTCGGATCCACTAGTAAC

GGCCGCCAGTGTGCTG

GAATTC

GGCTTCTATGTGCTGGGCTGCAGGTACTGGTGTGTGAACATGTTGAG

CTCACTGTCCAACCAACCGGCTTTATTCCTGAGCAGCTTTGCTTTGCTCTGAAGTGAGTCAAGAACCTC

AATTTTCTTGTTCATGGCGGCAATTTCCTGCTCCAGGTTCTCCCGGGTGACGTCAACTTGCTGACACAG

ATGAGCAAAGGTCCCAGACAGTTCCTGCTGGACTTGGTGGCTGCAGTTGGAGCCAGTGTAGCTGATGAC

AAGCTGCAGCTTCTCGCTGGCATGCTCCACAAACTGGCGCTTGAAGGCCCTCTCCTTGGCCTTGGTGGT

CCAGGTCAGACGCTCATAGACGTAGAGGAGGCCATAGAGCCCAAAGGAGAGGGCAATGAGCCGCCAGCC

CACTGCCTTCCACACCACTCCTCCAACAACAAGAATGCCCATGGAGGTCCTGGATGTCAAGGAGGCCAG

GCCGGTAACCATGGAAACCATGAACTCCTCCTGGGTGAGCGAGCCCTGTGGCAGTGGGGGCATGCTGGG

GTTGGCTGGCGTCAGAGGGATGGGACGCTGGACCTGGTCATTGTAACCCATCAAGGCCCGACGGCTGTT

CTTGGGGCCCAGGAACCTATTCACCAGCATGGTCCATCCGAGAGAGAAATGGAACTCAATGTCTTCCTG

GAAATCAAACCCNCTTGCACANAAGCCGAATTNTGAAATATTCATNACACTGGCGGCCGTTGAACATGC

NTTANAGGCCCAATTCCCTAAGGGAGTCTATTAAATTACTGGCCG

NCBI blast ID: 1018489490-12259-12970

>gi|7662003|ref|NM_014874.1| Homo sapiens mitofusin 2 (MFN2), mRNA Length =

4550

Identities = 664/666 (99%)

Query: 4 ctatgtgctgggctgcaggtactggtgtgtgaacatgttgagctcactgtccaaccaacc 63 |||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2596 ctatctgctgggctgcaggtactggtgtgtgaacatgttgagctcactgtccaaccaacc 2537 Query: 64 ggctttattcctgagcagctttgctttgctctgaagtgagtcaagaacctcaattttctt 123 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2536 ggctttattcctgagcagctt tgctttgctctgaagtgagtcaagaacctcaattttctt 2477 Query: 124 gttcatggcggcaatttcctgctccaggttctcccgggtgacgtcaacttgctgacacag 183 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2476 gttcatggcggcaatttcctgctccaggttctcccgggtgacgtcaac ttgctgacacag 2417 Query: 184 atgagcaaaggtcccagacagttcctgctggacttggtggctgcagttggagccagtgta 243 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2416 atgagcaaaggtcccagacagttcctgctggacttggtggctgcagttggagccagtgta 2357

Query: 244 gctgatgacaagctgcagcttctcgctggcatgctccacaaactggcgcttgaaggccct 303 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2356 gctgatgacaagctgcagcttctcgctggcatgctccacaaactggcgcttgaaggccct 2297 Query: 304 ctccttggccttggtggtccaggtcagacgctcatagacgtagaggaggccatagagccc 363 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2296 ctccttggccttggtggtccaggtcagacgctcatagacgtagaggaggccatagagccc 2237 Query: 364 aaaggagagggcaatgagccgccagcccactgccttccacaccactcctccaacaacaag 423 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2236 aaaggagagggcaatgagccgccagcccactgccttccacaccactcctccaacaacaag 2177 Query: 424 aatgcccatggaggtcctggatgtcaaggaggccaggccggtaaccatggaaaccatgaa 483 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2176 aatgcccatggaggtcctggatgtcaaggaggccaggccggtaaccatggaaaccatgaa 2117 Query: 484 ctcctcctgggtgagcgagccctgtggcagtgggggcatgctggggttggctggcgtcag 543 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2116 ctcctcctgggtgagcgagccctgtggcagtgggggcatgctggggttggctggcgtcag 2057 Query: 544 agggatgggacgctggacctggtcattgtaacccatcaaggcccgacggctgttcttggg 603 |||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||| Sbjct: 2056 agggatgggacgctggacctggtcattgtagcccatcaaggcccgacggctgttcttggg 1997 Query: 604 gcccaggaacctattcaccagcatggtccatccgagagagaaatggaactcaatgtcttc 663 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1996 gcccaggaacctattcaccagcatggtccatccgagagagaaatggaactcaatgtcttc 1937 Query: 664 ctggaa 669 |||||| Sbjct: 1936 ctggaa 1931

6.

Expression of mitofusin1/GTPase in NSCLC determined by RT-PCR

Amplified DNA fragments were extracted from the gel and sequence determined.

90% of NSCLC overexpressed Mfn1 as determined by RT-PCR. However, there was

no extraneous band appeared below the authentic fragment, and therefore no aberrant

splicing of Mfn1 gene was detected in the tumor nest.

7.

Subcloning of mitofusin1/GTPase gene into an expression vector, and to deliver

recombinant DNA into bacteria to express recombinant mitofusin1/GTPases.

Mf1S gene translate Protein sequence

：

Theoretical pI: 8.51 Mw (average mass): 22933.10

1 11 21 31 41 51 | | | | | | 1 MRGSHHHHHH GSDDDDKLPR SLASTPTAPT TPATPDNAKE ELMITLVTGL ASVTSRTSMG 60 61 IIIVGGVIWK TIGWKLLSVS LTMYGALYLY ERLSWTTHAK ERAFKQQFVN YATEKLRMIV 120 121 SSTSANCSHQ VKQQIATTFA RLCQQVDITR KQLEEEIARL PKEIDQLEKI QNNSKLLRNK 180 181 AVQLENELEN FTKQFLPSSN EES

Mf1S sequence 中藍色部分為主要蛋白序列部份，紅色部分為包含表現載

體與設計之蛋白酵素切割部位(底線標示之 DDDDK 序列)所增加之序

列，預計表現蛋白質之大小為 23 kDa。

下圖：為 13% SDS-PAGE gel。

Control 組為未經 IPTG 誘導的菌體全蛋白。

#1 及#2 分別為兩株不同的菌株，經 1 mM IPTG 誘導 4 hrs 後之全

蛋白，在紅色箭頭所指區域有明顯的新增蛋白出現，分子量與所預

期 23 kDa 相吻合。

由表現結果看來，蛋白質表現已無問題，然而其表現量偏低，

需大量表現並進行純化進度及抗體之製備。