猪链球菌2型05ZYH33强毒株srt同源序列及srtA原核表达抗原性的分析

Homologous analysis of streptococcus suis srt gene and prokaryotic expression of srtA

目的阐明猪链球菌(S.suis)2型人源强毒力株05ZYH33基因组中srt的数量和分布,原核表达srtA,并分析表达产物的抗原性。方法借助生物信息学软件对05ZYH33基因组中Sortase及其相关家族蛋白进行同源性分析,克隆srtA基因,表达的重组蛋白用Western blot检测其抗原活性,制备Sortase鼠多抗血清,间接ELISA检测鼠多抗血清的滴度。结果通过系统的搜索和分析,发现05ZYH33基因组中含有6个Sortase的同源性序列,其中SrtA及Sortase-like归属sortase家族中的A组,SrtBCD和SrtE属B组。srtA基因在原核细胞中得到高效表达,表达产物可与S.suis2型的兔多抗血清发生特异性的反应。以重组蛋白为免疫原制备的多抗血清效价达1∶6400。结论我国S.suis2型强毒力株05ZYH33含有6个srt基因,与国外报道相比新发现一个sortase-like基因。srtA基因可在原核系统高效表达,重组蛋白具有良好的抗原活性,为进一步研究Sortase功能奠定了基础。

AIM： To elucidate the distribution of srt gene in the genome of S. suis 05ZYH33, prokaryotically express Sortase A and analyze its antigenicity. METHODS： Homologous genes encoding sortase family members were analyzed, and then the srtA gene of S. suis was cloned and sequenced. The recombinant protein was analyzed by SDS-PAGE and Western blot. The mice were immunized with recombinant protein and the antibody titer was determined by indirect ELISA. RESULTS： All the putative srt genes in the genome of 05ZYH33 were analyzed. Six genes were found to be homologous to srt of S. suis isolated in Japan. The predicted srtA and sortase-like proteins were members of Class A of sortase family while srtBCD and srtE belonged to Class B. Western blot analysis showed that the recombinant protein was reactive to with the serum from the rabbits infected with a virulent strain of S. suis Type 2. The antibody titer in blood serum reached 1：6 400 after immunization with recombinant protein four times. CONCLUSION： Compared with the isolated strain from Japan a new putative srt gene was found in 05ZYH33. The srtA gene was successfully expressed in prokaryotic system and the recombinant protein showed specific antigenicity, which is important for future research of the function of Sortase.