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.pdf

第33卷第3期
中山大学学报(医学科学版)
Vol33 No
2012年5月
JOURNAL OF SUN YAT-SEN UNIVERSITY (MEDICAL SCIENCES
May.2012
β溶血性链球菌htra基因的测序鉴定及表达賃白的
免疫反应性分析
倪琼琼,丁月霞,刘金来',余步云
(中山大学附属第三医院心血管内科,广东广州510630)
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hta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;将測序正确的A组链球菌(CAS)hta基因克隆至原核表达质粒pCEX4T-1,并在E, coli BL21中表达,应用
Western blot,以抗 CST 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hura基因与已知 GAS htra基因的一致性均达99%; Western blot显示,HrA融合蛋白可与抗 GST Rabbit mab以及
CAS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(CBS)、C组链球菌(GCS)htra序列不符;GAS感染动物后可产生抗
HrA蛋白抗体,可以认为HtrA为AS的显性免疫原,丽尚不能认为非A组链球菌中HtrA为其显性免疫原。
关键词:β溶血性链球菌;htra基因;克隆表达;免疫反应性
中图分类号:Q7
文献标志码:A
文章编号:1672-3554(2012)03-0351-06
Identification of htra Gene and Immunoreactivity Analysis of htra Protein in Beta
hemolytic Streptococci
N Qiong-qiong, DING Yue-xia, LIU Jin-lai, YU Bu-yun
( Deparment of Cardiology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China)
Abstract: 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 Ecoli BL21. The recombinant Hira 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 GS 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
Key words: beta-hemolytic streptococci; htra gene; protein expression; immunoreactivity
UJ SUN Yat-sen Univ(Med Sci), 2012, 33(3): 351-355
β溶血性链球菌(beta- hemolytic streptococci,体。按其C抗原,可分为18个组,对人致病的大多
BHS)是一类能够引起人或动物感染性疾病的病原属于A组链球菌( group A streptococcus,GAS)。GAS
收稿日期:2012-01-20
基金项目:广东省科技计划项目(2008B080701023
作者简介:倪琼琼,硕士研究生,E-mail:qiaoqiaoni0111163.com;*通信作者:刘金来,教授,主任医师,E-mail:liujing@hotmail.om,研
究方向:风心病疫苗