利用毕赤酵母菌SMD1168表达SSA重组抗原

Use of yeast cell Pichia pastoris SMA1168 to express the recombinant SSA antigen

目的利用电穿孔法将pPIC9k-SSA重组质粒导入毕赤酵母菌SMD1168真核表达系统进行诱导表达,获得SSA重组抗原。方法以人白血病淋巴细胞HL-60株为模板扩增SSA基因,将其转入pPIC9k质粒构建pPIC9k-SSA重组质粒,采用电穿孔法将其导入毕赤酵母菌SMD1168真核表达系统进行诱导表达,获取目的蛋白。结果成功构建pPIC9k-SSA重组质粒并导入毕赤酵母菌SMD1168真核表达系统中,获得稳定整合目的基因的菌株;重组菌株经诱导表达出产物相对分子量约为50kDa的SSA蛋白,具有抗原性可与SSA抗体特异性结合。结论本实验室通过分子克隆技术,从毕赤酵母菌SMD1168真核表达系统中获得了人SSA抗原,为今后建立斑点免疫金渗滤法(DIGFA)法检测抗SSA抗体奠定基础。

To obtain the recombinant SSA antigen by transferring the recombinant plasmid pPIC9k-SSA to yeast cell Pichia pastoris SMDl168 expression system for inductive expression by using electroporation technique, the SSA gene was amplified by using human leukemic lympboeytes as template and then transferred into plasmid pPICgk to construct the recombinant plasmid pPIC9k-SSA. By using the electroporation technique, the product was finally transformed into P. pastoris SMD1168 expression system for inductive expression to produce the target protein. In these ways, the recombinant plasmid pPICgk-SSA was successively constructed and it could be transformed into P. pastoris SMD1168 expression system to obtain a strain with stable integration of target gene. The SSA protein with relative molecular weight of 50 kDa was obtained from inductive expression of the recombinant strain. This protein exhibited antigenicity and could bind specifically with antibody against SSA. The results in this study could provide a solid basis for further development of the dot immunogold filtration assay （DIGFA） to detect SSA antibody.