猪链球菌2型分泌核酸酶(SsnA)的表达、ELISA方法建立及其在鉴别诊断中的应用  

Expression of Streptococcus suis Type 2 Secreted Nuclease A(SsnA),Development of the SsnA-ELISA and Its Application in the Differential Diagnosis

作　　者：朱剑[1] 康超[1] 张安定[1] 金梅林[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《畜牧兽医学报》2011年第2期228-235,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家973计划(2010CB534000);国家科技重大专项课题(2008ZX10004-013;2009ZX10602-14);转基因生物新品种培育重大专项(2009ZX08009-141B);国家公益性行业农业专业专项资助(200803018);湖北省自然科学基金(2009CDB171)

摘　　要：猪链球菌2型（Streptococcussuis type 2,SS2）是一种广泛分布的人畜共患病原菌,严重危害着人类健康和养猪业的发展。本试验旨在研究分泌核酸酶基因（SsnA）作为分子诊断标识在对SS2感染的鉴别诊断中所起的作用。作者以SS2-LT菌株基因组DNA为模板,通过PCR扩增SsnA基因片段,将该片段克隆到表达载体pET-28a（＋）中,在大肠杆菌BL21（DE3）中成功表达,获得大小为46 ku的融合蛋白,经SDS-PAGE和Western blot分析表明,重组蛋白质具有抗原反应活性。以纯化的融合蛋白作为包被抗原,建立了SsnA-ELISA鉴别诊断方法。对已知背景的人工感染及疫苗免疫血清和临床血清进行检测,比较感染血清和免疫血清的检测结果,结果发现两者差异显著（P〈0.05）,表明建立的SsnA-ELISA方法具有良好的检测效果。利用该方法对采集自8个省份的1 446份血清进行检测,显示SS2的阳性率为0%~14.77%,平均阳性率为5.39%。本研究结果表明：猪链球菌2型SsnA可以作为一个良好的鉴别感染的分子诊断标识;对临床血清的检测结果表明感染及流行与气候环境存在明显的关系。Streptococcus suis serotype 2（SS2） is a zoonotic infectious pathogen associated with a wide range of diseases in human health and pig industry.This article aims to study the role of SsnA gene as a molecular diagnosis marker in the differential diagnosis of SS2.SsnA gene was amplified using SS2-LT genome DNA as template and the sequence was then inserted into expression vector pET-28a（＋）.The plasmid was introduced into E.coli BL21（DE3） for expression and 46 kD recombinant protein was obtained,which had been proved to be immunoreactive by SDS-PAGE and Western blotting.An SsnA-ELISA diagnostic method was developed by using the purified SsnA fusion protein as coating antigen.The detected results of immune serum and infected serum were compared by using SsnA-ELISA Kit,and the significant difference（P0.05） presented indicates that the established SsnA-ELISA owns excellent detecting effect.1 446 serum samples collected from 8 provinces of China were then tested by SsnA-ELISA Kit and the result show that the positive rate ranged from 0%-14.77% and the average positive rate was 5.39%.Our study show that SsnA of Streptococcus suis serotype 2 could be used as a good molecular diagnosis marker in distinguishing infected or immunized animals;in addition,our clinical data indicates that there exists an evident relationship between the infection and epidemicity of SS2 and its climatic environment.

关 键 词：猪链球菌2型 分泌核酸酶(SsnA) 鉴别诊断 ELISA 

分 类 号：S852.611[农业科学—基础兽医学]