毛果杨丝氨酸蛋白酶抑制剂基因PtrSPI的克隆及真核表达

利用PCR技术从毛果杨(Populus trichocarpa)中分离得到1条丝氨酸蛋白酶抑制剂基因PtrSPI,该基因cDNA编码区全长为1176 bp,可编码391个氨基酸。BlastP预测结果显示,该丝氨酸蛋白酶抑制剂属于SERPIN超家族。多序列比对结果表明,毛果杨PtrSPI基因的氨基酸序列与已报道的胡杨(P.euphratica)丝氨酸蛋白酶抑制剂氨基酸序列XP_011030699同源性最高,相似性为92%,且蛋白的C端氨基酸序列都含有高度保守的反应中心环(RCL)。在此基础上,成功构建了PtrSPI基因的重组真核表达载体pPICK-PtrSPI并获得了重组毕赤酵母菌株GS115-PtrSPI。利用终质量分数为0.5%甲醇诱导重组真核蛋白表达,SDS-PAGE电泳结果显示,在42648.45处出现了重组蛋白,与预期值一致。

Cloning and Eukaryotic Expression of A PtrSPI Gene from Populus trichocarpa

A 1176 bp PtrSPI gene coded 391 aa was cloned from Populus trichocarpa by PCR.The BlastP results showed PtrSPI protein belongs to the SERPIN super family,and the multiple sequence alignment results show that PtrSPI amino acid sequence which contain a conservative amino acid sequences-reaction center loop(RCL)in c-terminal shared 92%identity with the serpin from P.euphratica.The PtrSPI gene was then constructed into vector pPIC9K to obtain the recombinant vector pPIC9K-PtrSPI.After the recombinant vector was transformed into P.pastoris strain GS115,the multicopy yeast transformants was induced by the final concentration of 5%methanol to obtain recombinant protein.Finally,the SDS-PAGE showed that recombinant proteins appeared at 42648.45,which was consistent with the expected values.