表达HIV-1 gag-gp120嵌合基因酵母工程菌的构建及表达条件的优化

目的：构建表达HIV-1gag-gp120嵌合基因的酵母工程菌，并优化表达条件。方法：将gag-gp120嵌合基因插入到酵母表达载体pHILS1中，构建了表达质粒pHILGP。线性化质粒电转化毕赤酵母菌GS115后进行整合，通过PCR及表达产物的SDS-PAGE和ELISA等方法筛选 阳性酵母工程菌，并优化表达条件。结果：成功构建表达融合蛋白的酵母工程菌，表达量为13%左右。表达蛋白能与HIV-1阳性血清发生反应，但其相对分子质量（Mr）比预计计算的值要小。最佳表达条件为：BMMY培养基，85%溶解氧，培养时间3d，1%甲醇诱导浓度。结论：表达的融合蛋白具有很好的抗原特异性，存在于上清中，这有利于目的蛋白的分离纯化。

Construction of engineering yeast strain expressing gag-gp120 chimeric gene of HIV-1 and optimization of the expression condition

Objective To make up an engineering yeast strain expressing HIV 1 gag gp120 chimeric gene. Methods gag gp120 chimeric gene was inserted into a yeast expression vector pHILS1 and the expression plasmid pHILGP was constructed. After the plasmids were linearized and electrotransformed into the yeast strain GS115, an engineering yeast strain was screened by PCR. SDS PAGE, ELISA analysis of expressed products, and the expression condition was optimized. Results An engineering yeast strain was successfully established. The amount of the expressed protein was approximately 13% of the soluble protein in the supernatant. The expressed protein could reacted with HIV 1 positive serum,but the relative molecular mass ( M r) of gag gp120 fusion protein was smaller than the expected value. Conclusion The expressed protein has good antigen specificity and is exist in the supernatant of the culture which favors isolation and purification of interest protein. [