两种新型HBsAg基因的毕赤酵母表达载体构建及其表达

目的：从乙肝患者血清中发现2个有突变的乙型肝炎病毒株，从中扩增出HBsAg(乙肝表面抗原)基因，构建酵母表达载体，并在巴斯德毕赤酵母中表达，以鉴定这2个基因表达产物的活性。方法：利用PCR技术从乙肝患者血清中扩增出乙肝表面抗原基因．并将其连接到pPIC3．5K中，转化毕赤酵母，通过甲醇诱导，利用SDS-PAGE和ELISA检测表达的蛋白。结果：通过序列测定结果表明成功地构建了毕赤酵母表达载体，聚丙烯酰胺凝胶电泳(SDS—PAGE)和ELISA证明这2个基因在毕赤酵母中能有效表达。且表达产物均具有生物活性。结论：从实验结果发现乙肝表面抗原决定簇以外的少数氨基酸变化不影响其活性。因此可以利用这2个基因对乙肝表面抗原进行表达。

Construction of Pichia pastoris Expressive Vectors of Two New Hepatitis B Surface Antigen Genes and Expression

Objective:We found two new viruses of hapatitis B from a suffer's serum,then abtained the genes of HBsAg by PCR,constructed the expressive vectors to express the HBsAg through the Pichia pastoris for assaying the activity.Method:We amplified the HBsAg genes by PCR,cloned the genes into the pPIC3.5K,transformed it to GS115,used SDS-PAGE and ELISA to assay the expressed proteins.Results:The expressive vectors we constructed were successful by assayed.And SDS-PAGE and ELISA after the methanol induced which indicate the proteins have biological activity.Conclusion:Few of amino acid mutation do not change the biological activity except the antigenic determinant.So we can use the two genes to express the active HBsAg.