体内表达新基因trag在猪链球菌的分布及其免疫反应性分析

Identification and detection of trag:a new infection-related gene expressed in vivo from isolates of Streptococcus suis

【目的】trag(transfer gene G)是利用IVIAT(in vivo induced antigen technology)通量筛选鉴定的猪链球菌2型(Streptococcus suis type 2,SS2)感染相关因子,研究该基因在猪链球菌(Streptococcus suis,SS)中的分布情况,研究康复血清与免疫血清在免疫印迹中的反应性有无,间接证明其在体内感染与体外培养时表达差异。【方法】鉴于我国分离株trag与GenBank公布的SS2北美株89/1591的trag序列有95.8%的同源性,据此设计和合成一对检测引物,对SS2我国江苏及四川流行株、其他临床分离株和参考株及SS1、SS1/2、SS9、SS7及C群猪源链球菌共43株进行PCR扩增。另设计一对引物,扩增5株SS代表菌株trag的完整阅读框,并对扩增产物进行测序。据软件分析后,选择TRAG(Transfer protein G)免疫原性良好的区域片段的核酸设计表达引物,PCR扩增后定向克隆至表达载体pET28a(+)构建表达质粒,表达蛋白转印到PVDF膜上,分别与SS2猪康复血清和猪高免血清反应。【结果】trag在SS2中94%(30/32)阳性,SS9中67%(4/6)阳性,SS7阳性,SS1、SS1/2及C群菌阴性。5株细菌TRAG的氨基酸序列与SS2中国株98HAH33、05ZYH33及北美株89/1591同源性>97%。所获得重组蛋白只能被康复血清识别。【结论】从SS致病株中检出感染相关基因trag,提示该基因可能与SS致病性有关,重组蛋白的免疫转印结果表明,TRAG可能与SS2体内感染相关。

[Objective] The trag （transfer gene G） was one of the novel infection-related factors identified by in vivo-induced antigen technology （IVIAT） from Streptococcus suis type 2 expression libraries with swine convalesecent sera in our former research. We detected the distribution of trag in different Streptococcus suis isolates and identify the differential expression of the new infection-related factor between in vivo and in vitro condition. [Methods] According to the sequence of trag of North American strain 89/1591, a pair of primers were designed to detect the distribution of trag in total 43 SS isolates. Another pair of primers were designed to amplify the ORF of trag of 5 SS representive strains （ZY05719, HA9801, 98012, SH040805, SH040917）. Partial gene of trag was cloned and inserted into expression vector pET28a（＋）, and induced by IPTG to express recombinant TRAG. The recombinant protein was probed with swine convalescent sera and immune sera respectively. [Results] The trag was detected in the most of SS2 isolates （30/32）, in SS9 isolates （4/6）, and I isolate of SS7, while it was not found in SS2 European strain ATCC43765, avirulent strain SS2 T15,1 isolates of SS1, 1 isolates of SS1/2 and 2 isolates of group C streptococcal strains from pigs. Comparisons between the sequences of TRAG of 5 isolates with that of SS isolates, showed a high homology （〉97%） with North American strain 89/1589 and China strains 98HAH33,05ZYH33. The immunoreactivity was only presented with convalescent sera. [Conclusion] The trag was detected from virulent SS isolates but not from avirulent strain, which suggested that this gene may be re- lated to the pathogenicity of SS. The special reactivity was only present with convalescent sera, and it indicated that TRAG might play a role during SS2 invasive course.