大肠杆菌融合基因K88ac-STⅡ的减毒鼠伤寒沙门疫苗菌的构建与表达

Construction of a recombinant live attenuated salmonella typhimurium vaccina strain carrying K88ac-STⅡ fusion eusion gene of enterotoxigenic Escherichia coli

本研究为构建表达产肠毒素性大肠杆菌融合基因K88ac-STⅡ的无抗性减毒鼠伤寒沙门氏菌疫苗株,从质粒pET-K88ac-STⅡ中扩增编码融合蛋白K88ac-STⅡ的基因并插入线性T载体pBS-T中,经酶切、PCR、测序鉴定正确后,再用限制性内切酶切下,插入含有asd基因的表达载体pYA3334,构建pYA-K88ac-STⅡ重组质粒。重组质粒鉴定正确后电穿孔转入缺失腺苷酸环化酶基因(△cya)、环腺苷酸受体蛋白基因(△crp)以及天冬氨酸β-半醛脱氢酶基因(△asd)的鼠伤寒沙门菌(X4550)中,以获得重组菌株X4550(含pYA-K88ac-STⅡ)。结果显示该无抗药性的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,口服该疫苗菌株无明显的毒性作用。通过本试验成功构建了表达K88ac-STⅡ融合基因平衡致死的减毒鼠伤寒沙门重组菌X4550(含pYA-K88ac-STⅡ)。为防治仔猪腹泻提供了口服活菌疫苗候选株。

To construct a live oral Salmonella typhimurium vaccine strain expressing K88ac-ST Ⅱ fusion protein of enterotoxigenic Escherichia coli. The K88ac-ST Ⅱ gene was amplified by PCR from the plasmid pET-K88ac-ST Ⅱ and subcloned into pYA3334 containing asd gene. The resultant plsmid pYA-K88ac-STⅡ was transformed by electroporation into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain X4550. The plasmid was stable in the host under culture conditions and the expressed K88ac-ST Ⅱ could react positively with the anti-K88ac antiserum. The S, typhimurium X4550 （containing pYA- K88ac-ST Ⅱ ） strain could be further explored as a new live oral vaccine candidate against diarrhoea.