猪链球菌2型次黄嘌呤核苷酸脱氢酶缺失株的构建

 【目的】猪链球菌2型（Streptococcus suis type 2，SS2）为一种重要的人兽共患病病原，次黄嘌呤核苷酸脱氢酶（IMPDH）为SS2新近鉴定的一个蛋白，本文旨在体外构建基因缺失株明确IMPDH与SS2致病力的相关性。【方法】选用自杀性质粒，通过体外构建具有同源臂的重组自杀性质粒，电转化进入SS2，经同源重组，获得impdh基因缺失株。通过接种不同敏感动物，明确缺失株致病力的变化。【结果】经过PCR鉴定，impdh基因被氯霉素（cat）选择标记基因表达盒所代替，Western blot鉴定结果显示，IMPDH单抗没有检测到相应的IMPDH蛋白。SS2-H（△IMPDH）对6种糖的发酵能力降低、生长速度减慢、产酸减少一个pH滴度和对本动物和试验动物的致病力减低。SS2-H（△IMPDH））对于Balb/c小鼠LD50是8.3×108 CFU，而SS2-H的LD50是3.36×108 CFU;对新西兰家兔只引发体温升高(高于正常体温至少1℃)，不致死;对于断奶仔猪，只表现临床症状，但不引起死亡。【结论】本研究成功获得IMPDH缺失株，即SS2-H（△IMPDH），并且明确IMPDH的缺失导致了SS2对仔猪、家兔和Balb/c小鼠的致病力降低。

Construction and Identification of Streptococcus suis Type 2 Deficient in IMPDH Expression

【Objective】 Streptococcus suis type 2 (SS2) is an important zoonotic pathogen. The inosine 5-monophosphate dehydrogenase is a newly found protein of SS2, therefore, the objective of this study is to construct the impdh-knockout strain for further research on the correlation impdh with virulence. 【Method】 The suicide vector pSET4s and chloromycetin (cat) were used to construct the mutant of IMPDH gene by homologous recombination. 【Result】 The results from PCR and Western-blot methods showed that IMPDH gene was successfully replaced. Also, the SS2-H (△IMPDH) was found to be less competent for six carbohydrates metabolism, slower growth curve, far less acidic substances and be less virulent in mouse, rabbit and porcine models of infection than parent SS2. For Balb/c mice, the LD50 of SS2-H (△IMPDH) was 8.3×108 CFU, whereas the LD50 of SS2-H was 3.36×108 CFU. All rabbits infected with SS2-H (△IMPDH) of equal to or above 1.0×109 CFU presented only hyperthermy (i.e. at least 1℃ over the normal body temperature).All pigs infected with SS2-H (△IMPDH) presented only clinical syndromes and could not cause death. 【Conclusion】 The impdh-knockout SS2 was successfully constructed and named SS2-H (△IMPDH). The experiments pathogenicity to swine, Balb/c mice and rabbits indicate that SS2-H (△IMPDH) weakens virulence to sensitive animals.