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当前位置: 首页 > 文章 > 2型猪链球菌SspA基因序列及其免疫原性分析 南方农业学报 2015,46 (7) 1310-1314
Position: Home > Articles > Sequence and immunogenicity of SspA gene from Streptococcus suis type 2 Journal of Southern Agriculture 2015,46 (7) 1310-1314

2型猪链球菌SspA基因序列及其免疫原性分析

作  者：
胡巧云;袁小宁;熊毅;何奇松;黄胜斌;孙翔翔;吴军;颜健华;冯淑萍
单  位：
广西大学生命科学技术学院;广西动物疫病预防控制中心
关键词：
2型猪链球菌;SspA基因;分段表达;免疫原性
摘  要：
【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。
译  名：
Sequence and immunogenicity of SspA gene from Streptococcus suis type 2
作  者：
HU Qiao-yun;YUAN Xiao-ning;XIONG Yi;HE Qi-song;HUANG Sheng-bin;SUN Xiang-xiang;WU Jun;YAN Jian-hua;FENG Shu-ping;Li Jun;GUO Jian-gang;Animal Disease Prevention and Control Center of Guangxi;College of Life Sciences and Technology,Guangxi University;
关键词：
S.suis 2;;SspA gene;;segmented expression;;immunogenicity
摘  要：
【Objective】In 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(Ssp A) 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 Ssp A gene. The corresponding fragments were amplified from 112 clinical isolates, then Ssp A gene was sequenced and analyzed. Meanwhile, three expression plasmid(p ET28a-Ssp N, p ET28 aSsp M and p ET28a-Ssp C) containing Ssp A gene were transferred into Escherichia 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., p ET28a-Ssp N,p ET28a-Ssp M and p ET28a-Ssp C were induced with IPTG to express 3 fusion proteins. And the recombinant proteins Ssp N and Ssp C existed in supernatant of lysates, the recombinant protein Ssp M existed in precipitation of lysates. The molecular sizes of recombinant proteing Ssp N, Ssp M and Ssp C were 66, 65 and 32 k D, 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 react with nactivated serum. Besides, the reaction of Ssp N was the strongest among all reactions. 【Conclusion】The upstream sequence of Ssp A gene is more conservative, and the Ssp N protein expressed by E. coli has stronger immunogenicity,which has potential for detecting and immunizing S. suis 2. Therefore Ssp A gene can be used as a candidate antigens molecular marker for diagnosis of S. suis 2.

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