蜂毒肽/变构hIL-2融合蛋白的原核表达及其对宿主菌生长的影响

Prokaryotic Expression of Melittin/hIL-2 Mutant Fusion Protein and Its Effect on Growth of Host E. coli

目的构建蜂毒肽(Melittin)与变构hIL-2融合基因原核表达质粒,并检测表达的融合蛋白对宿主菌E.coli生长的影响。方法以质粒pGEX-4T-2/Melittin-IL-2(88Arg)为模板,通过PCR定点诱变为Melittin-IL-2(88Arg,125Ala),将PCR产物和pET-15b载体分别经双酶切后连接,构建重组表达质粒pET-15b/M-IL-2(88Arg,125Ala),转化E.coliBL21(DE3),IPTG诱导表达。SDS-PAGE及ELISA分析表达产物;检测诱导不同时间重组菌的A600值,绘制生长曲线,并进行活菌计数。结果PCR扩增的目的片段长542bp,重组表达质粒测序分析证明目的基因如预期突变;ELISA可检测到目的蛋白表达,但表达量较低;SDS-PAGE分析未见目的条带;诱导表达4h,重组菌A600值由0.8下降至0.6;诱导2h,活菌计数由108个/ml降至104个/ml。结论已成功构建了原核表达质粒pET-15b/M-IL-2(88Arg,125Ala),表达的融合蛋白可能对宿主菌具有毒性,从而杀伤宿主菌。

Objective To construct a prokaryotic expression vector for melittin / hIL-2 mutant fusion gene and determine the effect of expressed protein on growth of host E. coli. Methods A mutant Melittin-IL-2 （^88Arg,125Ala）was obtained through site-directed mutagenesis by PCR using plasmid pGEX-4T-2 / Melittin-IL-2（^88Arg）as a template. Both the PCR product and vector pET-15b were digested with BamHⅠand NdeⅠthen linked,and the constructed recombinant plasmid pET-15b / M-IL-2 （^88Arg,125Ala） was transformed to E. coli BL21（DE3）for expression under induction of IPTG. The expressed product was analyzed by SDS-PAGE and ELISA. The A600 of recombinant E. coli after induction for various hours were determined,based on which a growth curve was plotted. Meanwhile,the recombinant E. coli was subjected to viable counting. Results The target gene fragment at a length of 542 bp was amplified by PCR. Sequencing result proved that the target gene was mutated as expected. Low expression of target protein was proved by ELISA. However,no band of target protein was observed on SDS-PAGE profile. After induction for 4 h,the A600 of recombinant E. coli decreased from 0. 8 to 0. 6. After induction for 2 h,the viable count of in each milliliter of recombinant E. coli culture decreased from 10^8 to 10^4. Conclusion Recombinant plasmid pET-15b / M-IL-2（^88Arg,125Ala）was successfully constructed. The expressed fusion protein showed a potential toxicity which might kill the host E. coli.