β溶血性链球菌htra基因的测序鉴定及表达蛋白的免疫反应性分析  

Identification of htra Gene and Immunoreactivity Analysis of htrA Protein in Beta-hemolytic Streptococci

作　　者：倪琼琼[1] 丁月霞[1] 刘金来[1] 余步云[1] 

机构地区：[1]中山大学附属第三医院心血管内科,广东广州510630

出　　处：《中山大学学报（医学科学版）》2012年第3期351-356,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省科技计划项目(2008B080701023)

摘　　要：【目的】了解广州市儿童致咽炎β溶血性链球菌(BHS)中htra同源基因的分布并初步探讨其表达蛋白在BHS中的免疫反应性。【方法】运用PCR扩增从患儿分离的44株BHS的htra基因并测序,测序结果与NCBI中已知基因序列进行同源性比对;将测序正确的A组链球菌(GAS)htra基因克隆至原核表达质粒pGEX4T-1,并在E.coli BL21中表达,应用Western blot,以抗GST Rabbit mAb鉴定重组融合蛋白表达,以BHS免疫小鼠抗血清检测目的蛋白的免疫反应性。【结果】44株BHS的htra基因与已知GAS htra基因的一致性均达99%;Western blot显示,HtrA融合蛋白可与抗GST Rabbit mAb以及GAS免疫小鼠血清特异性结合,而非A组链球菌及对照组免疫血清则呈阴性结果。【结论】从患儿分离的BHS均携带与GAS相同的htra基因,这与基因库中已知的B组链球菌(GBS)、C组链球菌(GCS)htra序列不符;GAS感染动物后可产生抗HtrA蛋白抗体,可以认为HtrA为GAS的显性免疫原,而尚不能认为非A组链球菌中HtrA为其显性免疫原。[ Objective] To investigate whether htra homologous genes were present in the 44 BHS isolates from children with acute throat infection or tonsillitis in Guangzhou recently and analyze the immunoreactivity of htra protein. [ Methods ] Htra genes were amplified by polymerase chain reaction （PCR） and identified by sequencing. Then the htra gene from GAS was cloned into pGEX4T-1 vector and HtrA protein was expressed in E.coli BL21. The recombinant HtrA protein was identified by Western blot with anti-GST rabbit mAb and its immunoreactivity was analyzed by Western blot with the sera from mice infected with BHS. [Results] All of the 44 BHS isolates harboured the htra genes which were 99% identical with the gene of GAS. Western blot confirmed that both anti-GST rabbit mAb and the sera from mice infected with GAS could react specifically with the recombinant HtrA protein, while the sera from other group could not. [ Conclusions ] All of the BHS isolates contain htra gene which is the same as the known GAS gene and is different from the known GBS and GCS gene. GAS infection is able to induce antibody against HtrA protein. This indicates that HtrA protein acts as a dominant immunogen in GAS, while the immunoreactivity of HtrA proteins from other groups of streptococci is still unknown.

关 键 词：Β溶血性链球菌 htra基因 克隆表达 免疫反应性 

分 类 号：Q7[生物学—分子生物学]