单核细胞增生性李斯特菌溶血素基因的原核表达

目的为获得大量的单核细胞增生性李斯特氏菌（Listeria monocytogenes，Lmo）溶血素（Hemolysin，hty）蛋白，以便研制Lmo诊断试剂及其在疫苗研制方面的作用。方法本文应用Primer Premier5．00设计引物，引入13amHI和x幻工酶切位点。以前期合成构建的pMDl8-T-hly质粒为模板，通过PCR方法扩增出Lmo0586株溶血素基因。相应酶切后，克隆到原核表达载体pGEX-6p-1中，构建pGEX-6p-hly重组质粒，转化入大肠杆菌BL21（DE3）进行表达。带有重组质粒pGEX-6p-hly的大肠杆菌BL21（DE3）经IPTG诱导后，进行SDS-PAGE及免疫印记分析。结果PCR体外扩增hly基因产物大小约为1624bp，成功构建了重组表达质粒pGEX-6p—hly；SDS-PAGE显示蛋白表达带的分子量约为72ku，重组蛋白主要以包涵体形式表达。表达量占菌体总蛋白的20．8％；Western免疫印记表明具有良好的反应原性。结论在国内首次构建重组质粒pGEX-6p-hly，并以融合蛋白的形式进行了高效表达，同时该蛋白具有特异的抗原反应性，为研制Lmo诊断试剂及其在疫苗研制中的作用奠定了基础。

Prokryotic expression of the hemolysin gene from Listeria monocytogenes

To obtain large amounts of Hemolysin(hly) protein of Listeria monocytogenes to be used for laboratory diagnosis and preparation of vaccine for this infection,the hly DNA fragment was gained from plasmid pMD18-T-hly with a pair of primers containing Bam HI and Xho I sites by PCR.After digestion with restriction enzymes and purification,the PCR products were cloned into prokaryotic expression vector pGEX-6p-1 and then transformed to E.coli BL21(DE3) cells to express the best characterized determinant hemolysin gene of L.monocytogenes.The prokaryotic expression of hemolysin gene was induced by IPTG and analyzed by SDS-PAGE and Western blotting.It was found that the size of amplified hemolysin gene was demonstrated to be 1624 bp in length,and the correct recombinant plasmid pGEX-6p-hly was subsequently constructed and expressed under the induction of IPTG.The expressed product was found to be 72 ku in molecular weight.As demonstrated by SDS-PAGE,the recombinant proteins were produced mostly in form of inclusion bodies,consisting 20.8% of the total cellular proteins,and these proteins exhibited excellent reactivity with antibodies against L.monocytogenes.It is concluded that fusion protein was obtained through the recombinant expression vector pGEX-6p-hly,and this protein can be highly expressed in E.coli with excellent reactivity with antibodies against L.monocytogenes.The results of this work provide a foundation for further studies on the laboratory diagnosis and preparation of vaccines for this infection.