以减毒沙门氏菌为载体的GM-CSF与生长抑素DNA疫苗的安全性和稳定性

Safety and stability of an oral fusion-expression plasmid of GM-CSF and SS using attenuated Salmonella as carrier

将含GM-CSF基因与生长抑素（SS）的融合真核表达质粒pGM—CSF／SS转入减毒沙门氏菌株CS022。选择40只22～24g的雌性小鼠，随机分成4组，其中1组为对照组，口服生理盐水；2～4组为试验组，分别用上述重组减毒沙门氏菌株CS022以10^8、10^9、10^10CFU的剂量口服免疫小鼠，2周后以相同的剂量加强免疫，研究pGM-CSF／SS真核表达质粒在体内的稳定性和减毒沙门氏菌对小鼠的安全性。同时通过体外传代，研究该真核表达质粒在体外的稳定性。结果表明：10^8、10^9CFU剂量口服小鼠，成活率达100％，而10^10CFU剂量口服小鼠成活率降低至90％，腹泻率达50％。体外传10代和口服免疫4周后从小鼠肝脏和脾脏分离的减毒菌用琼脂糖凝胶电泳和酶切鉴定法证实，减毒菌中的重组质粒在体外和侵入小鼠体内过程中具有较好的稳定性。质粒DNA在传10代以后，虽然每代的质粒DNA含量有些变化，但总体趋势基本是恒定的，第10代时，质粒DNA的含量与第1代的含量差异不显著。上述结果提示，利用该减毒沙门菌作为载体传递pGM—CSF／SSDNA疫苗免疫小鼠具有相对安全性和稳定性，为进一步有效激发机体的免疫应答提供了前提。

Fusion-expression plasmid （pGM-CSF/SS） of GM-CSF and SS was transformed into attenuated Salmonella CS022. A total of 40 female mice aged 22 days old, weighted at 20 24 g were divided radomly into 4 groups to examine the safety and stability of the vaccine in vivo and in vitro. All mice of 4 groups were given twice immunizations at an interval of 2 weeks by oral administration. Group 1 as control was immunized with 0. 85 % salt solution. Group 2-4 as experimental groups were orally inoculated witha dose of 10^8,10^9,10^10CFU per mouse, respectively. The results showed that the survivor rate of group 2 and group 3were 100%, while that of group 3 reduced to 90%. Diarrheal rate of group 3 was 50%. The bacteria isolated from the liver and spleen of mice taken 4 weeks post-inoculation （in vivo） and after 10 generation cultured （in vitro） were identified by agarose gel electrophoresis and restriction enzyme analysis for Salmonella. The results indicated that the strain pGM-CSF/SS could invade into the mice and the recombinant plasmid was stable both in vivo and in vitro culture. So the oral pGM-CSF/SS using attenuated Salmonella as carrier possess relatively safety and stability, which provide foundation for effective immune response.