畜牧兽医学报 ›› 2011, Vol. 42 ›› Issue (2): 228-235.doi:

• 预防兽医 • 上一篇    下一篇

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

朱剑,康超,张安定,金梅林*   

  1. 华中农业大学 农业微生物学国家重点实验室,武汉 430070
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2011-02-25 发布日期:2011-02-25
  • 通讯作者: 金梅林

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

ZHU Jian, KANG Chao, ZHANG An-ding, JIN Mei-lin*   

  1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University,Wuhan 430070, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2011-02-25 Published:2011-02-25

摘要: 猪链球菌2型(Streptococcus suis 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。

Abstract: 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 (P<0.05) 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.