本文選題：豬CR1-like　切入點：生物信息學　出處：《山西農(nóng)業(yè)大學》2015年碩士論文

【摘要】：目的：利用巴斯德畢赤酵母(Pichia pastoris)表達系統(tǒng),對豬紅細胞CR1-1ike的補體調(diào)控蛋白區(qū)域(complement control protein domain, CCP)進行串聯(lián)表達,為進一步深入研究豬CR1-like的作用機制奠定基礎(chǔ)。方法：對豬CR1-like的CCPs氨基酸序列進行生物信息學分析,篩選出CCPs的基因序列,密碼子優(yōu)化后人工合成CCPs基因序列；將合成的基因序列連入表達載體pwPICZalpha中,利用Pichia pastoris對其進行重組表達, SDS-PA GE和Western blotting檢測表達上清；利用Ni-NTA Resin柱和強陰離子交換柱對重組蛋白進行純化；采用激光共聚焦顯微鏡觀察重組蛋白與經(jīng)兔血清致敏菌株或未致敏菌株的免疫粘附反應(yīng),及豬CRl-like單克隆抗體先與重組蛋白反應(yīng),再與豬紅細胞共孵育后的免疫阻斷反應(yīng)。結(jié)果：生物信息學分析結(jié)果顯示,豬CRl-like基因含有19個CCP,并選定3-6和8-11兩段CCPs基因序列進行表達；SDS-PAGE和Western blotting結(jié)果顯示,表達上清中存在所預(yù)期的目的蛋白,說明重組酵母構(gòu)建成功；表達上清經(jīng)純化濃縮后,獲得了分別濃度為0.828 mg/mL和0.945mg/mL的兩種重組蛋白；免疫熒光結(jié)果顯示,重組蛋白可與致敏短小芽孢桿菌發(fā)生粘附,并能夠阻斷CRl-1ike單克隆抗體與豬紅細胞的結(jié)合。結(jié)論：本試驗成功獲得兩株可穩(wěn)定遺傳表達豬CR1-like CCPs的Pichia pastoris菌株；重組蛋白表現(xiàn)出補體結(jié)合活性,具有類免疫粘附受體功能。
[Abstract]:Aim: to express the complement control protein domain (CCPs) of porcine erythrocyte CR1-1ike by using Pichia pastoris expression system. Methods: the CCPs amino acid sequence of porcine CR1-like was analyzed by bioinformatics, the gene sequence of CCPs was screened out, and the CCPs gene sequence was synthesized after codon optimization. The synthesized gene sequence was inserted into the expression vector pwPICZalpha and expressed by Pichia pastoris. The expression supernatant was detected by SDS-PA GE and Western blotting, and the recombinant protein was purified by Ni-NTA Resin column and strong anion exchange column. Laser confocal microscopy was used to observe the immune adhesion reaction between recombinant protein and strain sensitized or not sensitized by rabbit serum, and the monoclonal antibody against porcine CRl-like was first reacted with recombinant protein. Results: the results of bioinformatics analysis showed that the porcine CRl-like gene contained 19 CCPs, and selected 3-6 and 8-11 segments of CCPs gene sequence to express SDS-PAGE and Western blotting. The expression of the target protein in the supernatant indicated that the recombinant yeast was successfully constructed, and two recombinant proteins with concentrations of 0.828 mg/mL and 0.945mg/mL were obtained after purification and concentration of the supernatant. The recombinant protein could adhere to the sensitized Bacillus pumilus and block the binding of CRl-1ike monoclonal antibody to porcine erythrocyte. Conclusion: in this experiment, two stable Pichia pastoris strains expressing porcine CR1-like CCPs were successfully obtained. The recombinant protein exhibits complement binding activity and has the function of immune adhesion receptor.
【學位授予單位】：山西農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：Q786;S852.4

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