重症肌无力单链抗体A7在毕赤酵母中的表达及检测

发布时间：2018-05-01 20:06

本文选题：重症肌无力 + 单链抗体　；参考：《延边大学》2007年硕士论文

【摘要】： 目的：将抗乙酰胆碱受体单链抗体(single chain variable fragment，ScFv)基因转化至毕赤酵母GS115，筛选阳性转化子并诱导蛋白表达，制备单链抗体蛋白。方法：从Ecoli．DH5α中提取目的基因并纯化，将电泳检查后确认正确的pPIC9K重组载体经过限制性核酸内切酶Sal I线性化处理，电穿孔导入甲醇营养型毕赤酵母GS115中，，提取酵母基因组DNA进行PCR，检测外源基因的插入情况。挑取拷贝数高的阳性转化子进行甲醇诱导表达，应用鼠抗c-myc单克隆抗体进行斑点杂交试验检查酵母培养上清液中单链抗体A7蛋白的表达情况。并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹试验(WesternBlotting)确认目的蛋白的分子量。结果：电泳检查发现了大小分别为10000 bp和9300 bp左右的重组载体pPIC9K-ScFvA7基因和真核表达载体pPIC9K基因片段；经PCR后电泳确定目的基因已整合到毕赤酵母染色体基因组中。经检测得知，诱导表达后的酵母培养液上清中有目的蛋白表达，而对照酵母培养液上清未发现目的蛋白的表达。在SDS-PAGE和蛋白印迹试验中发现一条分子量约为44KD的单一带。结论：成功将重组载体pPIC9K-ScFvA7整合到毕赤酵母染色体基因组中，使ScFvA7蛋白在毕赤酵母中成功表达，并对此基因的表达情况进行了初步鉴定，为下一步鉴定目的蛋白活性及特异性奠定了基础，也为今后单链抗体对重症肌无力的治疗和基因治疗准备了材料。
[Abstract]:Objective: single chain variable scFvv gene was transformed into Pichia pastoris GS115 to screen positive transformants and induce protein expression to prepare single chain antibody protein. Methods: the target gene was extracted from Ecoli.DH5 伪 and purified. The correct pPIC9K recombinant vector was confirmed by electrophoretic analysis. The recombinant vector was linearized by restriction endonuclease Sal I, and electroporated into methanol-nutritious Pichia pastoris GS115. The genomic DNA of yeast was extracted to detect the insertion of exogenous gene. The positive transformants with high copy number were selected for methanol induced expression. The expression of scFv A7 protein in the supernatant of yeast culture was detected by dot blot hybridization with mouse anti c-myc monoclonal antibody. The molecular weight of the target protein was confirmed by SDS-PAGE and Western blotting. Results: the recombinant vector pPIC9K-ScFvA7 gene and eukaryotic expression vector pPIC9K gene were found to be about 10000 BP and 9300 BP, respectively, and the target gene was integrated into the genome of Pichia pastoris by PCR electrophoresis. The results showed that the expression of target protein was found in the supernatant of yeast culture medium, but not in the supernatant of control yeast medium. A single band with a molecular weight of about 44KD was found in SDS-PAGE and Western blot. Conclusion: the recombinant vector pPIC9K-ScFvA7 was successfully integrated into the genome of Pichia pastoris and the ScFvA7 protein was successfully expressed in Pichia pastoris. It lays a foundation for the further identification of the activity and specificity of the target protein, and also provides materials for the treatment and gene therapy of myasthenia gravis by scFv in the future.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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