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目的构建和表达细粒棘球绦虫重组卡介苗(BCG)菌株rBCG-EgG1Y162。方法通过基因工程技术将细粒棘球绦虫抗原EgG1Y162的编码基因与大肠埃希菌(E.coli)-分枝杆菌穿梭表达质粒载体pMV361重组,并转化E.coli后进行扩增。重组质粒pMV-EgG1Y162经PCR和双酶切鉴定后,进行测序。将鉴定正确的rpMV-EgG1Y162通过电穿孔技术转化至感受态BCG菌株中,构建rBCG-EgG1Y162。经PCR和双酶切鉴定正确后,扩增培养2周,并于45℃放置30 min,诱导目的蛋白表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析蛋白表达情况,并以兔抗原核表达重组蛋白EgG1Y162血清为一抗进行蛋白质印迹(Western blotting)分析。结果重组质粒rpMV-EgG1Y162经PCR扩增和双酶切后,均获得约360 bp的EgG1Y162目的基因片段,与预期片段长度一致,测序结果表明插入序列正确。将其通过电穿孔转化BCG菌株后,rBCG-EgG1Y162生长良好,经酶切和PCR鉴定正确。SDS-PAGE和Western blotting结果显示,目的表达产物的相对分子质量(Mr)约为71 000。结论构建和表达了细粒棘球绦虫rBCG-EgG1Y162菌株。
Objective To construct and express the recombinant BCG bacterium BCG (rBCG-EgG1Y162). Methods The gene encoding EgG1Y162 of Echinococcus granulosus was recombined with shuttle vector pMV361 of E. coli-Mycobacterium by gene engineering and transformed into E. coli for amplification. The recombinant plasmid pMV-EgG1Y162 was identified by PCR and double enzyme digestion, then sequenced. The rpMV-EgG1Y162 strain was transformed into competent BCG strain by electroporation to construct rBCG-EgG1Y162. After identified by PCR and restriction enzyme digestion, the cells were expanded and cultured for 2 weeks and then incubated at 45 ℃ for 30 min to induce the expression of the target protein. The protein expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The recombinant protein EgG1Y162 was expressed as a primary antibody against rabbit antigens. Western blotting analysis was performed. Results After the recombinant plasmid rpMV-EgG1Y162 was amplified by PCR and double-digested, about 360 bp of EgG1Y162 gene fragment was obtained, which was consistent with the expected fragment length. The sequencing results showed that the inserted sequence was correct. After transfection of BCG strains by electroporation, rBCG-EgG1Y162 grew well and was identified by restriction enzyme digestion and PCR. The results of SDS-PAGE and Western blotting showed that the relative molecular mass (Mr) of the expressed product was about 71,000. Conclusion The echinococcus granulosus rBCG-EgG1Y162 strain was constructed and expressed.