表达核小体Th表位减毒鼠伤寒沙门菌株的构建

Construction and expression of the attenuated Salmonella typhimurium strain expressing the nucleosome Th epitope

目的:构建表达核小体Th表位(H2B14-28)的口服减毒鼠伤寒沙门菌株。方法:将H2B14-28基因插入原核表达载体pYA3149中,构建重组质粒pH2B(pYA3149-H2B14-28)后,转入鼠沙门菌株X4550,斑点印迹鉴定表位肽的表达。观察X4550(pH2B)体内外携带重组质粒的稳定性。结果:酶切鉴定和基因测序显示pH2B构建成功。斑点印迹证实表位肽的表达。重组菌株在体内、外营养选择压力下,可较稳定地携带重组质粒传代繁殖。结论:成功构建了能稳定表达核小体Th表位的可口服减毒鼠伤寒沙门菌株,为进一步直接应用减毒鼠伤寒沙门菌活菌苗在肠道内表达抗原诱导SLE免疫耐受奠定基础。

Objective： To construct a live oral vaccine of attenuated Salmonella typhimurium strain expressing the nucleosome Th epitope （H2B14-28）. Methods： H2B14-28 gene was inserted into the prokaryotic expression vector pYA3149 by gene recombination technique. The resulting recombinant plasmid pH2B （pYA3149-H2B14-28） was transferred into the attenuated S, typhimurium vaccine strain X4550 and got the live vaccine strain X4550 （pH2B）. The expression of the peptide was detected with Dot blotting. Stability of pH2B in the strain X4550 was studied under in vitro and in vivo condition. Results： Restriction analysis and DNA sequence analysis showed that the pH2B was successfully constructed. The peptide could be detected by Dot blotting. The recombinant plasmid pH2B was stable in the X4550 strain under in vitro and in vivo condition. Conclusion＂ The prokaryotic expression vector expressing nucleosome Th epitope in live attenuated S. typhimurium strain is successfully constructed, which may lay a foundation for using the live oral vaccine to test the SLE immumotolerance.