猪红细胞CR1-like基因CCPs串联表达及体外活性研究

发布时间：2018-03-23 12:57

本文选题：猪CR1-like　切入点：生物信息学　出处：《山西农业大学》2015年硕士论文

【摘要】：目的：利用巴斯德毕赤酵母(Pichia pastoris)表达系统,对猪红细胞CR1-1ike的补体调控蛋白区域(complement control protein domain, CCP)进行串联表达,为进一步深入研究猪CR1-like的作用机制奠定基础。方法：对猪CR1-like的CCPs氨基酸序列进行生物信息学分析,筛选出CCPs的基因序列,密码子优化后人工合成CCPs基因序列；将合成的基因序列连入表达载体pwPICZalpha中,利用Pichia pastoris对其进行重组表达, SDS-PA GE和Western blotting检测表达上清；利用Ni-NTA Resin柱和强阴离子交换柱对重组蛋白进行纯化；采用激光共聚焦显微镜观察重组蛋白与经兔血清致敏菌株或未致敏菌株的免疫粘附反应,及猪CRl-like单克隆抗体先与重组蛋白反应,再与猪红细胞共孵育后的免疫阻断反应。结果：生物信息学分析结果显示,猪CRl-like基因含有19个CCP,并选定3-6和8-11两段CCPs基因序列进行表达；SDS-PAGE和Western blotting结果显示,表达上清中存在所预期的目的蛋白,说明重组酵母构建成功；表达上清经纯化浓缩后,获得了分别浓度为0.828 mg/mL和0.945mg/mL的两种重组蛋白；免疫荧光结果显示,重组蛋白可与致敏短小芽孢杆菌发生粘附,并能够阻断CRl-1ike单克隆抗体与猪红细胞的结合。结论：本试验成功获得两株可稳定遗传表达猪CR1-like CCPs的Pichia pastoris菌株；重组蛋白表现出补体结合活性,具有类免疫粘附受体功能。
[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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q786;S852.4

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