牛支原体P81蛋白的表达及纯化

发布时间：2018-04-08 07:45

  本文选题：牛支原体　切入点：P蛋白　出处：《中国病原生物学杂志》2017年01期

【摘要】：目的构建3种牛支原体P81蛋白原核表达质粒,分析其在大肠埃希菌中的表达情况,并纯化P81蛋白。方法根据GenBank发表的牛支原体P81基因序列(GenBank登录号:AY627040.1),密码子优化后合成P81全基因序列。构建原核表达质粒pET28a-P81、pET32a-P81和pGEX-6P-1-P81,经IPTG诱导后采用SDS-PAGE电泳分析P81蛋白在原核表达系统中的表达情况;应用Ni-NTA对目的蛋白进行纯化。结果pET28a-P81、pET32a-P81和pGEX-6P-1-P81均高效表达分子质量单位为78ku左右的P81蛋白。表达产物进行Ni-NTA亲和层析,得到纯化的P81蛋白。结论构建的重组原核表达质粒pET28a-P81、pET32a-P81、pGEX-6P-1-P81能在大肠埃希菌中高效表达牛支原体P81蛋白,为进一步分析该蛋白的抗原性奠定了基础。
[Abstract]:Objective to construct three prokaryotic expression plasmids of Mycoplasma bovis P81 protein, analyze its expression in Escherichia coli, and purify P81 protein.Methods according to the P81 gene sequence of Mycoplasma bovis published by GenBank and GenBank accession number: AY627040.1, the whole P81 gene sequence was synthesized after the codon was optimized.The prokaryotic expression plasmids pET28a-P81, pGEX-6P-1-P81 and pGEX-6P-1-P81 were constructed. The expression of P81 protein in prokaryotic expression system was analyzed by SDS-PAGE electrophoresis after induction by IPTG, and the target protein was purified by Ni-NTA.Results both pET28a-P81 pET32a-P81 and pGEX-6P-1-P81 expressed P81 protein with 78ku or so.The purified P81 protein was obtained by Ni-NTA affinity chromatography.Conclusion the recombinant prokaryotic expression plasmid pET28a-P81 pET32a-P81 pGEX-6P-1-P81 can efficiently express Mycoplasma Bovis P81 protein in Escherichia coli, which lays a foundation for further analysis of the antigenicity of the protein.
【作者单位】： 军事医学科学院军事兽医研究所;广西大学动物科学技术学院;吉林农业大学动物科技学院;吉林大学动物医学学院;延边大学;
【基金】：吉林省科技发展计划项目(No.20140309024NY,20140623019TC,20160623024TC)
【分类号】：S852.62
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本文编号：1720698