人甲状旁腺激素(1-34)二联体与人血清白蛋白融合蛋白的构建及表达

Construction and Expression of Recombinant Fusion Protein hPTH(1-34)a'a-HSA

构建人甲状旁腺激素(1-34)二联体与人血清白蛋白融合蛋白的表达载体,并表达得到该融合蛋白。通过设计强特异性的引物,利用重叠PCR技术,定向定量的拼接得到hPTH(1-34)二联体-HSA融合蛋白的基因;将构建好的融合基因插入表达载体pPIC9K,大量扩增重组质粒,并用SalI线性化,电击转化毕赤酵母GS115,经组氨酸缺陷和G418抗性双重筛选得到阳性转化子;挑选阳性转化子进行甲醇诱导表达。测序结果表明得到的重组质粒pPIC9K-hPTH(1-34)二联体-HSA与目标设计完全一致;基因组PCR鉴定结果证明成功构建了hPTH(1-34)二联体-HSA融合基因的毕赤酵母(GS115)表达系统;SDS-PAGE电泳表明融合蛋白获得了表达,尿微量白蛋白试剂盒测定甲醇诱导表达3d后融合蛋白的产量为127 mg/L。

It was to Construct expression vector of the gene of recombinant fusion protein hPTH （ 1-34）.-HSA and obtain the fusion protein. Two hPTH（1-34） gene with different 5＇ and 3＇tails were obtained from the plasmid pPIC9K-PTH（1-84）-HSA by overlapping PCR technology. Then, the concatenate and HSA was fused by the same technology. The fused gene including HAS gene was cloned into vector pPIC9K. The plasmid pPIC9K-hPTH （1-34） a,a -HSA was linearized, and transformed into GS115 strain of pichia pastoris by electroporation. Then,the recombinant strains were screened by G418 resistance. The PC R results showed that the plasmid pPIC9K-hPTH （1-34）a,a, -HSA was constructed and transformed into GS115 successfully. Fusion protein can express and the production was 127 mg/L induced by methanol for 3 days.