乙型肝炎病毒PreS2S基因在毕赤酵母中表达的研究

构建重组质粒PHIL-D2/PreS2S以研究乙肝病毒PreS2(120-146)S基因编码蛋白在毕赤酵母中的表达。通过PCR扩增获得PreS2S片段,插入含AOX1启动子的Pichia Pastoris表达载体PHIL-D2中,构建重组表达质粒PHIL-D2/PreS2S,转化酵母宿主菌GS115。挑取阳性克隆摇床培养,甲醇诱导表达。通过ELISA、RPHA鉴定表达产物。成功构建了PHIL-D2/PreS2S真核表达载体,经过序列分析,插入的基因为在中国流行的adr亚型。在毕赤酵母中重组载体表达了S蛋白,S蛋白的表达量为34.9 mg/L,PreS2抗原检测为强阳性。利用毕赤酵母表达系统能够有效地表达乙型肝炎病毒的PreS2S蛋白,PreS2S蛋白具有良好的生物学活性。

Expression of HBV PreS2S Gene in Pichia pastoris

In order to construct recombinant plasmid PHIL-D2/PreS_2S and express HBV PresS_2S gene in Pichia pastoris,the PreS_2S gene amplified by PCR from PMD18T/S_2S was inserted into the P.pastoris expression vector PHIL-D2 containing AOX1 promoter.The recombinant expression plasmid PHIL-D2/PreS_2S was constructed and transformed into GS115.The positive transformants were induced by methanol for the expression of the PreS_2S gene.Products after the induction were analyzed with ELISA and RPHA.The results showed that recombinant plasmid of PHIL-D2/PreS_2S has been constructed successfully and sequence analysis suggested that the inserted foreign gene belong to adr subtype,which is the epidemic type in China.The PreS_2S gene has been expressed successfully in P.pastoris.Expression quantity of S protein is 34.5 mg/L and PreS_2 antigen is strongly positive.Therefore,PreS_2S gene has been expressed successfully with P.pastoris vector.And the PreS_2S protein in the test has good bioactivity.