Identification Of Htra Gene And Immunorectivity Analysis Of HtrA Protein In Beta-hemolytic Streptococci

【Objective】Beta-hemolytic streptococci(BHS) is a pathogen causing both human andanimal infections. Group A streptococcus(GAS) can couse disease ranging fromsuperficial infections of the throat or skin to highly invasive and potentially lethalinfections,including Infection sequela such as allergic reactions glomerulonephritisand Rheumatic heart disease.While Group B、C、G streptococcus were first knownas pathogens cause animal disease, in recent years, They have becomeincreasingly recognized as pathogens causing a number of human infections thatclinically resemble infections due to GAS. Horizontal genetic transfer within andamong the different serogroups of BHS has also been reported，genetic transferand selective pressure may result in the emergence of virulent human pathogenicNon-GAS strains with the potential to cause diseases classically associated withGAS. High temperature requirement A (HtrA) was first described as a housekeeping protease, HtrA-like proteases are involved in the virulence of gram-negative andgram-positive pathogens,mainly by their role in resistance and survival,such as to resist thermal or oxidativestress. But whether htra homologous genes are present in all the BHS isolates andthe immunorectivity of HtrA protein have not been clear.In this study, we investigated whether the htra homologous genes were presentin the44BHS isolates from children with acute throat infection or tonsillitis inGuangzhou recently, and cloned htra gene into plamid for constructing theprokaryotic expression plamid. And the combinant proteins were expressed. Theexpression of protein HtrA was confirmed by Western blot analysis, mice wereinjected with all bacteria vaccine, and the immunorectivity of HtrA protein wasanalysed. This provide the foundation to explore the immunity to infection of BHSand the research on diagnostic reagent and vaccine.【Method】1．We conducted daily prospective surveillance of school children aged4to15years with pharyngitis or tonsillitis in Guangzhou, collected pharyngeal swabbing todetermine the prevalence of BHS carriage. htra gene was amplified by PCR andsequenced,then compared to the the DNA sequence in NCBI.2．Htra gene was amplified by PCR. The gene was digested with BamHI, XhoI,and then cloned into prokaryotic expression plamid pGEX4T-1.The protein HtrA wasinduced by IPTG and expressed in E.coli BL21. The recombinant HtrA protein wasidentified by Western blot with anti-GST rabbit mAb.3．BALB/c mice were randomly individed into5groups, and immunizedrespectively with inactive GAS,GBS,GCS,GGS and PBS as control. Sera frommouse was obtained before and after immuinizatiao. The immunorectivity of HtrAprotein was analysed by Western blot with the sera from mice infected with BHS. 【Result】1．All of the44BHS isolates harboured the htra genes which were99%identical with the gene of GAS.2．Htra gene was amplified by PCR, after sequencing cloned into pGEX4T-1.Prokaryotic expression plamid pGEX4T-1/htra was successfully constructed.3．The recombinant HtrA protein was expressed in E.coli BL21as inclusionbody. The experiment results of Westernblot showed the protein expressed canreact specifically with anti-GST rabbit mAb and the recombinant HtrA-GST proteinwas comfirmed.4． Western blot confirmed that the sera from mice infected with GAS couldreact specifically with the recombinant HtrA protein, while the sera from other groupcouldn’t.【Conclusion】1．All of the BHS isolates contain htra gene which is the same as the knownGAS gene and is different from the known GBS and GCS gene.2．GAS infection is able to induce antibody against HtrA protein. This indicatesthat HtrA protein acts as a dominant immunogen in GAS, while the immunorectivityof HtrA proteins from other groups of streptococci is still unknown.