表达pCDR1 Th表位减毒鼠伤寒沙门菌株的构建

目的构建表达pCDR1 Th表位的可口服减毒鼠伤寒沙门菌株。方法全基因合成pCDR1两条核苷酸链，在C端加上6-His标签，退火互补后插入原核表达载体pYA3149中，构建重组质粒pR1，将该重组质粒转入鼠沙门菌终宿主菌株X4550，获得杂合菌株X4550（pR1）。体外诱导表达后斑点印迹鉴定表位肽的表达。结果酶切鉴定和基因序列测定显示重组质粒构建成功。体外诱导后6h细菌裂解上清液中，检测到表位肽的表达，该蛋白能与鼠抗His单克隆抗体特异结合。重组菌株在体外营养选择压力下，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pCDR1 Th表位的可口服减毒鼠伤寒沙门菌株。

Construction and expression of the attenuated Salmonella typhimurium strain expressing pCDR1 Th epitope

Objective To construct a attenuated Salmonella typhimurium live oral vaccine strain expressing pCDR1 Th epitope.Me-(thods Duplicate) bands of pCDR1 were synthesized and 6-His tag was added at C terminal.After annealing and complementation,the product was inserted into the prokaryotic expression vector pYA3149 by gene recombination technique.The resulting recombinant plasmid pR1 was transferred into the attenuated S.typhimurium vaccine strain X4550 and the live vaccine strain X4550(pR1) was obtained.The expression of the peptide was detected with dot blot assay.Results Restriction enzyme analysis and DNA sequence analysis showed that the pCDR1 Th epitope had been successfully inserted into pYA3149.The peptide could be detected in the split supernatant of the bacteria 6 h after IPTG induction.This peptide could specifically bind to mouse 6-His monoclonal antibody.The recombinant plasmid pR1 was stable in the X4550(pR1) strain under culture condition.Conclusion The prokaryotic expression vector expressing pCDR1 Th epitope in live attenuated Salmonella typhimurium is successfully constructed.