乳酸乳球菌介导日本血吸虫重组LL-Sj26GST疫苗的构建、鉴定及表达

Construction,identification and expression of Lactococcus lactis_based recombinant LL-Sj26GST vaccine of Schistosoma japonicum

目的构建以乳酸乳球菌(LL)为载体的日本血吸虫(Sj)rLL-Sj26GST疫苗,并研究其表达效率。方法以pGEX-Sj26GST为模板PCR扩增Sj26GST基因片段,经XbaⅠ和HindⅢ双酶切后插入pMG36e,构建重组质粒pMG36e-Sj26GST,转化大肠埃希菌BL21(DE3)感受态细胞。抽提质粒,经双酶切鉴定正确后电穿孔转化乳酸乳球菌MG1363株,构建日本血吸虫rLL-Sj26GST疫苗,抽提质粒进行PCR鉴定。结果pMG36e-Sj26GST经双酶切获得预期大小的目的片段,重组质粒构建正确。提取具有罗红霉素抗性的rLL质粒进行PCR,扩增出676 bp的Sj26GST基因片段;SDS-PAGE分析显示rLL可表达26 ku的Sj26GST蛋白,表达蛋白约占菌体总蛋白的18%;Western blot检测表达蛋白可被Sj感染者血清识别。结论成功构建日本血吸虫rLL-Sj26GST疫苗,表达的Sj26GST蛋白具有反应原性。

Objective To construct Lactococcus lactis(LL)_based recombinant LL-Sj26GST vaccine of Schistosoma japonicum and observe its expression efficiency.Methods Sj26GST gene was obtained by PCR from the template of pGEX-Sj26GST,the gene was cloned into pMG36e to construct pMG36e-Sj26GST and transformed into E.coli BL21(DE3)competent cell,the recombinant plasmid was identified by restriction endonuclease digestion,then was electroporated into LL to construct rLL-Sj26GST vaccine,the vaccine was identified by PCR.Results The products of restriction endonuclease digestion were the same as expected;PCR showed that 676 bp Sj26GST gene was amplified when the extracted plasmid from rLL as template;the relative molecular mass(Mr)of the expressed Sj26GST protein was approximately 26 ku by SDS-PAGE,the amount of the expressed protein was 18%of the total bacterial proteins;western blotting suggested that the expressed protein could be recognized by sera from Sj patients.Conclusion The rLLSj26GST vaccine was successfully constructed,and the expressed protein shows specific antigenicity.