摘要目的 构建日本血吸虫重组双歧杆菌属两歧双歧杆菌(Bifidobacterium bifidum,Bb)pGEx-Sj14-3-3疫苗,并进行鉴定.方法 从日本血吸虫成虫中提取总RNA,通过RT-PCR扩增Sj14-3-3抗原编码基因.将Sj14-3-3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-Sj14-3-3.用重组质粒将大肠埃希菌BL21(DE3)转化为感受态细胞,抽提重组质粒进行双酶切,鉴定载体片段和基因片段长度:采用电穿孔法,用重组质粒pGEX-Sj14-3-3转化Bh,构建Bb(pGEX-Sj14-3-3)疫苗,抽提疫苗的质粒进行PCR鉴定,比较重新构建的pGEX-Sj14-3-3基因片段长度与日本血吸虫成虫中Sjl4-3-3基因片段长度是否相同.结果 RT-PCR扩增出的日本血吸虫成虫Sj14-3-3基因片段长度为399 bp;双酶切鉴定,重组质粒pGEX-Sj14-3-3载体片段长度为4947 bp,Sj14-3-3基因片段长度为399 bp;在重组的Bb(pGEX-Sj14-3-3)疫苗中,得到Sj14-3-3基因片段长度为399 bp,与预期的结果一致.结论 成功构建了日本血吸虫重组Bb(pGEX-Sj14-3-3)疫苗.

abstractsObjective To construct and identify recombinant vaccine Bifwlobacterium bifidum(Bb)pGEX-Sj14-3-3 of Schistosoma japonicum(Sj). Methods Total RNA was extracted from adult Sj, antigen encoding gene Sj14-3-3 was amplified by RT-PCR and cloned into Escherichia coli (E. coli)-Bb shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Sj14-3-3. The recombinant plasmid was transformed into E. coli BL21 (DE3).The plasmid was extracted and identified by using BamH I and EcoR I. Then pGEX-Sjl4-3-3 was electroporated into Bb to construct recombinant Bb (pGEX-Sj14-3-3) vaccine. The extracted plasmid of the recombinant Bb (pGEX-Sj14-3-3) vaccine was identified by PCR, and the size of the products was compared with Sj14-3-3 gene of adult worms.Results Sj14-3-3 of 399 bp in length was amplified by RT-PCR. The products were digested by BamH I and EcoR I , and the fragments length of plasmid pGEX-Sj14-3-3 vector was 4947 bp, and of Sj 14-3-3 gene was 399 bp.The product of 399 bp Sj14-3-3 gene was also amplified by PCR from template of the extracted plasmid of the recombinant Bb(pGEX-Sj14-3-3 ) vaccine. The size of the product obtained was just the same as expected.Conclusion The recombinant Bb(pGEX-Sj14-3-3) vaccine of Sj is successfully constructed.