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目的构建并鉴定日本血吸虫重组双歧杆菌属两歧双歧杆菌(Bb)(pGEX-Sj26GST-Sj32)疫苗。方法从本室保存的BL21(DE3)(pET28α-Sj26GST-Sj32)重组菌中抽提质粒pET28α-Sj26GST-Sj32,PCR扩增Sj26GST-Sj32融合基因,将该融合基因定向克隆到大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT中,构建重组质粒pGEX-Sj26GST-Sj32。将此重组质粒转化至大肠埃希菌BL21(DE3)感受态细胞中,抽提质粒并进行BamHⅠ及EcoRⅠ双酶切,鉴定载体片段及目的基因片段长度。用电穿孔法将重组质粒pGEX-Sj26GST-Sj32转化至Bb,构建重组Bb(pGEX-Sj26GST-Sj32),抽提质粒,进行PCR鉴定。结果 PCR成功扩增出长度为1 991bp的Sj26GST-Sj32融合基因;双酶切证实Sj26GST-Sj32融合基因成功插入质粒pGEX-1λT中;PCR证实从重组Bb(pGEX-Sj26GST-Sj32)疫苗中扩增出1991bp的Sj26GST-Sj32融合基因。结论成功构建重组Bb(pGEX-Sj26GST-Sj32)疫苗。
Objective To construct and identify a recombinant Bifidobacterium bifidum (Bb) (pGEX-Sj26GST-Sj32) vaccine against Schistosoma japonicum. Methods Plasmid pET28α-Sj26GST-Sj32 was extracted from the BL21 (DE3) (pET28α-Sj26GST-Sj32) recombinant bacterium stored in our laboratory and the Sj26GST-Sj32 fusion gene was amplified by PCR. The fusion gene was cloned into Escherichia coli- Bifidobacterium shuttle vector pGEX-1λT, the recombinant plasmid pGEX-Sj26GST-Sj32 was constructed. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, the plasmid was extracted and digested with BamH Ⅰ and EcoR Ⅰ to identify the length of the vector fragment and the target gene fragment. The recombinant plasmid pGEX-Sj26GST-Sj32 was transformed into Bb by electroporation to construct recombinant Bb (pGEX-Sj26GST-Sj32). The plasmid was extracted for PCR identification. Results The Sj26GST-Sj32 fusion gene was successfully amplified by PCR. The double-digested Sj26GST-Sj32 fusion gene was successfully inserted into the plasmid pGEX-1λT. PCR was confirmed by PCR amplification from recombinant Bb (pGEX-Sj26GST-Sj32) The 1991 bp Sj26GST-Sj32 fusion gene was generated. Conclusion The recombinant Bb (pGEX-Sj26GST-Sj32) vaccine was successfully constructed.