• 沒有找到結果。

豬胸膜肺炎放線桿菌纖毛蛋白ApfA單株抗體之製備及分析

N/A
N/A
Protected

Academic year: 2021

Share "豬胸膜肺炎放線桿菌纖毛蛋白ApfA單株抗體之製備及分析"

Copied!
3
0
0

加載中.... (立即查看全文)

全文

(1)

1

豬胸膜肺炎放線桿菌纖毛蛋白 ApfA

單株抗體之製備及分析

指導教授:楊文仁 博士 國立高雄大學生物科技研究所 學生:黃炯中 國立高雄大學生物科技研究所 摘要 豬胸膜肺炎放線桿菌是一種感染豬呼吸道的病菌,會引起豬出血性、壞死 性、纖維素性胸膜肺炎,常造成豬隻死亡,為一種高致死率的傳染病。目前對於 此菌附著到豬隻呼吸道的機制了解甚少。纖毛是細菌吸附呼吸道表皮細胞常見的 媒介,而豬胸膜肺炎放線桿菌的纖毛是由數以千計的結構蛋白組成,ApfA是構 成此菌纖毛的結構蛋白,分子量約15 kDa。本實驗的目的是製備出ApfA的單株 抗體,分析其特性並使用此單株抗體進行ApfA抗原決定位的探討。首先將本實 驗室先前製備出的ApfA-pcDNA質體純化並製成DNA疫苗,對BALB/c小鼠進行 三次免疫注射,在確定產生抗ApfA之抗體效價後,取出小鼠脾臟細胞與骨髓瘤 細胞的融合,經過ELISA及西方墨點法篩選有陽性反應的融合瘤細胞。然後將陽 性反應的融合瘤細胞以極度稀釋法單株化後,證實得到能分泌ApfA蛋白單株抗 體的融合瘤細胞。將此融合瘤細胞以腹腔注射使小鼠產生腹水,取出腹水進行抗 體亞型分析,結果顯示此單株抗體亞型主要以IgG1為主。接著以protein A純化出 腹水內的單株抗體,完成單株ApfA抗體之製備。此外,本實驗利用定點突變將 ApfA蛋白質的cysteine置換為glycine,製備出C63G、C132G及C63&132G三株 ApfA突變菌株,來分析cysteine對ApfA抗原決定位之影響。以西方墨點法分析單 株抗體對ApfA突變後之辨識情形,結果顯示,由於單株抗體對於大腸桿菌XL-1 blue蛋白產生交叉反應而影響抗原決定位之分析,有待更進一步純化突變之蛋白 質來加以確認。抑制試驗發現製備的ApfA單株抗體能抑制豬胸膜放線桿菌之生 長。綜合實驗結果,本實驗完成之單株抗體,將為豬胸膜肺炎放線桿菌感染機制 的研究及疫苗之研發,提供重要的研究工具並奠定良好的基礎。 關鍵字:豬胸膜肺炎放線桿菌、ApfA 蛋白質、單株抗體、纖毛蛋白

(2)

2

The preparation and analysis of monoclonal antibody

against the fimbriae protein ApfA of Actinobacillus

pleuropneumoniae

Advisor: Dr. Wen-Jen Yang

Institute of Biotechnology, National University of Kaohsiung Student: Chiung-Chung Huang

Institute of Biotechnology, National University of Kaohsiung ABSTRACT

Actinobacillus pleuropneumoniae is a kind of pathogen which can infect the

porcine respiratory tract and induce hemorrhagic, necrotic and fibrinous pleuropneumonia of swine. The pleuropneumonia is an infectious disease with high mortality rate and causes the death of swine. So far, it is still known limited about the mechanism of how does this pathogen adhere to the porcine respiratory tract. Fimbriae are the medium that the bacteria usually used to adhere to the epithelia of respiratory tract. The fimbriae of A. pleuropneumoniae are composed of thousands of structural protein ApfA, which molecular weight is approximately 15 kDa. The purpose of this study is the preparation and characterization of monoclonal antibody against ApfA and use the monoclonal antibody to analyze the epitopes of ApfA. The ApfA-pcDNA plasmid constructed previously in our laboratory was used as DNA vaccine to immunize BALB/c mice. The antibody titer against ApfA raised was confirmed after third immunization. The mice splenocytes were isolated and fused to myeloma cells. The positive hybridoma clones were confirmed by screening with ELISA and Western blot analysis. Furthermore, the positive clones were used to conduct the limiting dilution to make sure the positive reaction is produced by monoclonal hybridoma. The results show that the hybridoma can produce monoclonal antibody against ApfA was obtained. The hybridoma cells were injected into the abdomen cavity of mice through intraperitoneal administration to produce ascite fluid. The subtypes of monoclonal antibody were analyzed and the results showed that the major subtype is IgG1. The monoclonal antibody was purified from ascite fluid and the monoclonal antibody against ApfA was obtained. In addition, the cysteine residues of ApfA were replaced by glycine through site-directed mutagenesis method. Three

(3)

3

ApfA mutant strains, C63G, C132G, and C63&132G double mutant, were obtained to analyze the influence of cysteine in ApfA epitopes. Western blot analysis was used to analyze the monoclonal antibody against these three mutant strains. The results showed that there is cross reaction between E. coli XL-1 blue which interfere with the epitope analysis. It is necessary to purify these mutated proteins for further analysis. The growth inhibition assay revealed that the monoclonal antibody could inhibit the growth of A. pleuropneumoniae. In summary, the monoclonal antibody produced in this study could provide a crucial research tool and establish the good groundwork for

A. pleuropneumoniae infection mechanism research and vaccine development.

Keywords: Actinobcacillus pleuropneumoniae, ApfA protein, monoclonal

參考文獻

相關文件

You are given the wavelength and total energy of a light pulse and asked to find the number of photons it

好了既然 Z[x] 中的 ideal 不一定是 principle ideal 那麼我們就不能學 Proposition 7.2.11 的方法得到 Z[x] 中的 irreducible element 就是 prime element 了..

Wang, Solving pseudomonotone variational inequalities and pseudocon- vex optimization problems using the projection neural network, IEEE Transactions on Neural Networks 17

volume suppressed mass: (TeV) 2 /M P ∼ 10 −4 eV → mm range can be experimentally tested for any number of extra dimensions - Light U(1) gauge bosons: no derivative couplings. =>

We explicitly saw the dimensional reason for the occurrence of the magnetic catalysis on the basis of the scaling argument. However, the precise form of gap depends

For pedagogical purposes, let us start consideration from a simple one-dimensional (1D) system, where electrons are confined to a chain parallel to the x axis. As it is well known

The observed small neutrino masses strongly suggest the presence of super heavy Majorana neutrinos N. Out-of-thermal equilibrium processes may be easily realized around the

Define instead the imaginary.. potential, magnetic field, lattice…) Dirac-BdG Hamiltonian:. with small, and matrix