表达幽门螺杆菌黏附素保守区的减毒鼠伤寒沙门氏菌株的构建

Construction of the Attenuated Salmonella typhimurium Strain Expressing Helicobacter pylori Conservative Region of Adhesin Antigen

为构建表达幽门螺杆菌 (Hp)黏附素保守区 (AB)的无抗性减毒鼠伤寒沙门氏菌疫苗 ,采用缺失腺苷酸环化酶基因 (Δcya)、环腺苷酸受体蛋白基因 (Δcrp)以及天冬氨酸 β 半醛脱氢酶基因 (Δasd)的鼠伤寒沙门菌 (X40 72 )作为宿主 ,将编码AB的基因插入Asd+ 的组成型表达载体pYA2 48,通过两次转化引入宿主菌 ,构建了表达AB基因平衡致死的减毒鼠伤寒沙门重组菌X40 72 (pYA2 48 AB) ,采用桥联法ELISA测定X40 72 (pYA2 48 AB)培养上清液和裂解上清液中AB的抗原性 ,参照Meacock叙述的方法及重组菌生长曲线的测定来确定重组菌株的稳定性 ,通过C5 7BL 6小鼠口服测定半致死量来确定重组菌的安全性。成功构建了表达AB的减毒鼠伤寒沙门菌重组菌株S .typhimuriumX40 72 (pYA2 48 AB) ,桥联法ELISA测定表明重组菌X40 72 (pYA2 48 AB)培养上清中AB的含量高于菌体裂解液 ,重组菌pYA2 48 AB在没有选择压力的情况下培养 10 0代 ,随机挑选的重组菌全部都能生长 ,且在ELISA测定AB抗原时均显阳性。重组菌的生长曲线测定表明 ,X40 72 (pYA2 48)和X40 72 (pYA2 48 AB)的生长状态基本一致 ;口服重组菌株X40 72 (pYA2 48 AB) 1 0× 10 1 0 cfu .3 0d后 ,C5 7BL 6存活率仍为 10 0 %。

Abstract To construct a non-resistance and attenuated Salmonella typhimurium strain which expresses conservative region of adhesin(AB) of Helicobacter pylori(Hp). The AB gene was amplified by PCR and inserted into the expression vector pYA248 containing asd gene and was introduceded into the delta Cya,delta Crp,delta Asd attenuated Salmonella typhimurium strain by twice transformations, which is a balanced lethal recombinant. Bridged ELISA method was used to measure AB expressed in sonicate and culture supernatant. According to Meacock's way and growth curve, stability of the recombinant is evaluated. Semi-lethal capacity test was used to evaluate the safty of recombinant. Results showed S.typhimurium X4072(pYA248-AB) was constructed successfully, recombinant X4072(pYA248- AB) content of supernatant serum was higher than that of thallus lytic liquor confirmed by bridged ELISA, and after recombinant pYA248- AB cultured 100 generation without selection pressure, all the recombinant germ selected randomly can grow, and the AB antigen was positive by ELISA detection. The growth curve of the recombinant germ showed that the growth state of X4072(pYA248)and X4072(pYA248- AB)were coincidence on the whole, and the survival rate of C57BL/6 was still 100%, 30 days after taking X4072(pYA248- AB)1.0×10 10cfu. orally. Non-resistance S.typhimurium X4072(pYA248- AB) was constructed successfully. The recombinant plasmid was stable indicated by in vitro experiment. And the recombinant strain was safe confirmed by animal experiment.This live vaccine strain is worthy to be considered as a new live oral vaccine candidate against Hp infection.