单核细胞增生性李斯特菌srtA基因的克隆与原核表达

Cloning and Prokaryotic Expression of the srtA Gene from Listeria monocytogenes

为了探究从新疆发病绵羊中临床分离的单核细胞增多性李斯特菌(简称LM90SB2)srt A基因的特异性及其在原核表达质粒p ET32a中的表达,本研究利用PCR技术扩增LM90SB2的srt A基因,测序后并进行序列比对分析,将其克隆到原核表达质粒p ET32a中构成重组表达质粒p ET32a-srt A。重组质粒转化入大肠杆菌BL21(DE3)中,异丙基硫代-β-D-半乳糖苷(IPTG)诱导使其表达,SDS-PAGE电泳检测,同时采用Western-blotting对表达产物进行了鉴定。结果表明:Srt A蛋白在BL21(DE3)中大量表达,表达产物经SDS-PAGE电泳和Western-blotting检测分析其为1个分子量为47 ku的融合蛋白,与预期的大小相符合,成功的构建了重组质粒p ET32a-srt A,并使其在BL21(DE3)中获得了高效的表达。本研究为下一步研究其对李斯特菌致病性的影响及制备单克隆抗体奠定了基础。

To investigate the specificity of srtA genes of Listeda monocytogenes LM90SB2 clinical isolated from diseased sheep in Xinjiang and its expression in plasmid pET32a.The srtA gene was amplified from LM9OSB2 by PCR,sequenced and ligased with the prokaryotic expression plasmid pET32a.The recombinant expression plasmid pET32a-srtA transformed into E.coli BL21 （DE3）.The transformed pET32a-srtA/BL21 were induced with isopropyl-β-D-thiogalactopyranosid（IPTG）,and the predicted fusion protein was detected by SDS-PAGE and Western blotting.These results showed that the prokaryotic expression plasmid for LM90SB2 srtA gene was successfully constructed and effectively expressed in E.coli BL21 （DE3）,the recombinant protein had molecular weight approximately 47 ku.This study provides basis for the research on the influence of the pathogenicity of listeria and the production of the specific monoclonal antibodies.