牛支原体P81蛋白的表达及纯化

Prokaryotic expression and purification of P81 in Mycoplasma bovis

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。

Objectives To improve the prokaryotic expression of the P81 gene of Mycoplasma bovis with different vectors and to purify P81. Methods Sequences of P81 were synthesized in accordance with the sequences of the P81 gene of M.bovis in GenBank.The P81 gene was cloned and 3recombinant plasmids were constructed：pET28a-P81,pET32aP81,and pGEX-6P-1-P81.Purified P81 protein was obtained using affinity chromatography. Results After protein expression was induced with 1.0 mmol/L of IPTG,P81 was expressed by pET28a-P81,pET32a-P81,and pGEX-6P-1-P81.A large amount of purified P81 was obtained using Ni-NTA affinity chromatography. Conclusion Results indicated that the P81 gene of M.bovis was successfully expressed by three prokaryotic expression plasmids.The P81 protein was purified,providing agood basis for further analysis of its immunogenicity.