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目的利用昆虫杆状病毒表达系统表达肠道病毒71型VP1蛋白并对其进行纯化。方法首先通过化学合成法合成EV71 VP1基因,然后将EV71 VP1基因插入到供体质粒pFast Bac1中,构成重组质粒pFast Bac1-VP1,再转入JM190感受态细菌扩增,然后提取重组质粒pFast Bac1-VP1转入DH10BacTM感受态细胞,通过转座从而获得重组质粒bacmid-VP1,采用脂质体Cellfectin Reagent将重组质粒bacmid-VP1转染Sf9细胞。通过SDS-PAGE、Western blot法检测目的蛋白的水平,经镍离子亲和层析,纯化VP1蛋白。结果 SDS-PAGE及Western blot法检测获得蛋白的相对分子质量与预期结果一致。Bradford分析纯化后蛋白的浓度为70μg/m L,纯化度达90%。结论利用昆虫杆状病毒表达系统成功表达EV71 VP1蛋白。
Objective To express and purify VP1 protein of enterovirus 71 using insect baculovirus expression system. Methods The EV71 VP1 gene was synthesized by chemical synthesis. The EV71 VP1 gene was inserted into the donor plasmid pFast Bac1 to construct the recombinant plasmid pFast Bac1-VP1, which was then transformed into JM190 competent bacteria for amplification. The recombinant plasmid pFast Bac1- VP1 was transformed into DH10BacTM competent cells, and the recombinant plasmid bacmid-VP1 was obtained by transposition. The recombinant plasmid bacmid-VP1 was transfected into Sf9 cells by using lipofectamine Cellfectin Reagent. The target protein level was detected by SDS-PAGE and Western blot, and VP1 protein was purified by nickel ion affinity chromatography. Results The relative molecular mass of protein obtained by SDS-PAGE and Western blot was consistent with the expected results. Bradford analysis of the purified protein concentration of 70μg / m L, the degree of purification of 90%. Conclusion The EV71 VP1 protein was successfully expressed in insect baculovirus expression system.