2型猪链球菌SspA基因序列及其免疫原性分析

Sequence and immunogenicity of SspA gene from Streptococcus suis type 2

【目的】明确2型猪链球菌的枯草杆菌素样丝氨酸蛋白酶(Ssp A)基因序列特征及其原核表达重组蛋白的免疫原性,为2型猪链球菌病的诊断及疫苗研究奠定基础。【方法】针对Ssp A基因上、下游序列分别设计1对引物,从112株2型猪链球菌临床分离株中扩增其相应片段,并进行测序分析;同时以Ssp A基因的重组质粒p ET28a-Ssp N、p ET28a-Ssp M和p ET28a-Ssp C分别转化感受态大肠杆菌BL21(DE3)后经IPTG诱导表达,再以SDS-PAGE检测融合蛋白表达情况,Western blotting鉴定其免疫原性。【结果】Ssp A基因上游片段较保守,而下游片段变异区间较大。在IPTG诱导下,含有重组质粒p ET28a-Ssp N、p ET28a-Ssp M和p ET28a-Ssp C的菌株表达出3段融合蛋白,其中,Ssp N和Ssp C重组蛋白存在于破碎菌体的上清液中,而Ssp M重组蛋白以包涵体形式存在于沉淀中,其大小为:Ssp N重组蛋白66 k D,Ssp M重组蛋白65 k D,Ssp C重组蛋白32 k D。Western blotting鉴定结果显示,3段重组蛋白均能与2型猪链球菌感染血清发生反应,且以Ssp N重组蛋白反应最强烈,而与以全菌灭活苗制备的猪血清未发生特异性反应。【结论】2型猪链球菌Ssp A基因上游片段较保守,经大肠杆菌重组表达的Ssp N蛋白免疫原性较强,且具备鉴别2型猪链球菌感染与免疫的潜力,可作为研究猪链球菌病血清学诊断的候选抗原片段。

[Objective]tn order to lay the foundation for diagnosis and vaccine research of Streptococcus suis type 2 （S. suis 2 for short）, the present experiment was conducted to study S. suis 2 subtilisin-like serine protease（SspA） gene＇s sequence features and immunogenicity of recombinant protein expressed by prokaryote. [ Method ]One pair of primers were designed based on downstream and upstream of SspA gene. The corresponding fragments were amplified from 112 clinical isolates, then SspA gene was sequenced and analyzed. Meanwhile, three expression plasmid （pET28a-SspN, pET28a- SspM and pET28a-SspC） containing SspA gene were transferred into Eschenchia coli BL21 （DE3）. The recombinant strain was inducted with IPTG to express recombinant protein. The purified recombinant proteins were detected by SDS-PAGE and its immunogenicity was identified by Western blotting. [Result]The results showed that the upstream sequence was more conservative than downstream sequence with extensive variation. The recombinant plasmids viz., pET28a-SspN, pET28a-SspM and pET28a-SspC were induced with IPTG to express 3 fusion proteins. And the recombinant proteins SspN and SspC existed in supernatant of lysates, the recombinant protein SspM existed in precipitation of lysates. The molecular sizes of recombinant proteing SspN, SspM and SspC were 66, 65 and 32 kD, respectively. The results of Western blotting demonstrated that the 3 recombinant proteins could react with serum infected with S. suis 2, however, which couldn＇t re- act with nactivated serum. Besides, the reaction of SspN was the strongest among all reactions. [ Conclusion ]The upstream sequence of SspA gene is more conservative, and the SspN protein expressed by E. coli has stronger immunogenicity, which has potential for detecting and immunizing S. suis 2. Therefore SspA gene can be used as a candidate antigens molecular marker for diagnosis of S. suis 2.