Secretion expression of dengue type 2 virus E proteins with Pichia pastoris system was performed in this paper, which could provide foundation for mechanism research of the interaction between host mosquitos and dengue virus. First of all,the protein E gene of DENV-2 was cloned through RT-PCR, and the cloned gene was then linked with the expression vector pPICZaB. The correct recombinant expression clones were selected by the ways of enzyme digestion, PCR and sequence analysis. Secondly, the recombinant vector was transformed into yeast (stain KM71H) by way of LiCl transformation procedure;the multi-copy carrier (Muts type) was also identified after selection of antibody, PCR, and phenotype. Finally,Western-blotting was applied for the surveillance on the E protein expression as well as the optimum culture time. As a result,E protein was successfully expressed by the yeast system and the best culture time 96 h was also confirmed. The present study would provide material foundation for uncovering the molecular mechanism during the dengue virus infection on the mosquitoes or human kind.%为研究登革病毒与蚊虫相互作用的分子机制,本研究对DENV-2的E蛋白基因进行了毕赤酵母分泌表达.首先,经RT-PCR方法克隆DENV-2的E蛋白基因,将克隆的基因与表达载体pPICZaB连接以构建表达重组子;并经酶切、PCR、测序筛选实验鉴定正确重组子.然后,重组子经氯化锂法转化毕赤酵母菌KM71H获得转化子;经抗生素、表型鉴定、PCR筛选得到Muts型的多拷贝转化子.最后,经甲醇诱导基因表达,Western-blotting方法鉴定蛋白表达情况及最佳表达时间.本研究对DENV-2的E蛋白成功进行了分泌表达,并且确定其表达最佳发酵时间为96 h;为进一步研究登革病毒与蚊虫相互作用的分子机制奠定了基础.
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