【作者】 孙长江；

【导师】 韩文瑜；

【作者基本信息】 吉林大学 ， 预防兽医学， 2008， 博士

【摘要】 本研究对分布于吉林省不同地区的9个大型养殖场的临床健康猪群中猪链球菌进行分子流行病学调查,采集样品774份,以PCR方法检出猪链球菌血清型1型、2型、9型和其它猪链球菌121株,阳性率15.63%,其中2型占28.93%,分离到猪链球菌112株,研究了猪链球菌的耐药性和猪链球菌2型分离株的致病性。以小鼠、斑马鱼和提前断乳豚鼠研究了猪链球菌2型CVCC606的致病性,并制备猪抗CVCC606高免血清,亲和层析获得的免疫相关抗原对5倍LD50剂量的猪链球菌2型CVCC606攻击小鼠的保护率为60%,而全菌抗原保护率为80%。以串联质谱法(MS/MS)鉴定免疫相关抗原,得到磷酸丙酮酸水合酶等5个具有重要意义的候选抗原。磷酸丙酮酸水合酶原核表达产物对猪链球菌2型CVCC606攻击小鼠保护率为50%。更多还原

【Abstract】 Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen which causing not only great economical losses in pig breeding industry, but also threatening the public health and food safety directly. In order to clarify the clinical health of swine carried Streptococcus suis and the problems existed in immunization for Streptococcus suis ,a molecular epidemiology study of were carried out to investigate the carrying rate of Streptococcus suis in post weaning piglets, growing pig, fatten pigs and sow herd of nine large farm distributed in the different area of Jilin province. 774 samples of nasal swab were collected. To detect of Streptococcus suis , the 16S rRNA gene was amplified by PCR method, 121 positive samples were detected out of all 774 samples, and the positive rate was 15.63%. The presence of Streptococcus suis in all farms were detected, However, there were significantly differences between the farms in positive ratio. Except the post weaning pig herd in farm H, the Streptococcus suis were detected in samples of every growth stage pig herd of all the farms.A multiplex PCR method was used to detect the most pathogenic Streptococcus suis serotype 1、1/2、2、7、9, and serotype 1、2、9 were detected from 774 samples. The serotype 2 was the most frequent (28.93%) serotype in positive samples, and no serotype 1/2 or 7strain was detected, untyping Streptococcus suis strains account for 57.02%.Including 28 strains Streptococcus suis type 2, 112 strains were identified as Streptococcus suis by bacteriology isolate method in total 774 samples.. Experimental models of mice were used to evaluate the virulence of the 18 SS 2 isolates, there were only 7 isolates were identified as pathogenic strains out from the 18 isolates. Genes of mrp and epf were detected from all the isolates of the SS2 including 7 isolates were positive with newly reported candidates PAI sequence of 89 kb.The studies of the drug resistance of 112 Streptococcus suis isolates showed that most of the Streptococcus suis exhibite multiple drug resistance with high level of resistance to erythromycin, tetracycline and sulfa drugs and a moderate level of resistance to amikacin, gentamicin and ciprofloxacin. The ratio of SS 2 resistant to Ampicillin, tetracycline, erythromycin were higher than that of all Streptococcus suis isolates, as for the proportion of drug resistant to other drugs of SS 2 was similar or lower than the overall isolates.In this experiment, SS2 CVCC606 strain which originally isolated from piglets suffering meningitis was introduced as the conference strain to study the immune related antigens. the virulence of CVCC606 strain were determined with BALB/c mice and zebra fish and early weaned guinea pigs. CVCC606 strain could not been detected in the brain tissue after infectious to the mice, zebra fish and early weaned guinea pig , But some of the brain tissue in infected mice showed inflammatory, Under the meningeal ,there were hyperaemia and congestive in early weaned guinea pigs. Compared with the other strains of SS 2, CVCC606 strain was confirmed as a moderate high virulence strain. This study paved way for screening immunoprotective antigen and research the protection of SS 2.In this experiment, the region(421-1700 bp) of epf gene that has not yet been studied presently was expressed with pGEX-3X vector and recombinant protein GST-TEF was purified. The results of immunoprotection test showed that protection rate of GST-TEF on BALB/c mice challenged with CVCC 606 strain at 5 folds of the LD50 was only 40%. But when the mice was immunized with recombinant protein of GST-TMRP and GST-EF , the protection rate of 62.5% was obtained.It is indicated that immunization of mice with GST-TEF enhance the immune protection of the GST-TMRP.To purify the immune related antigen of CVCC 606 strain and study the composition and immunoprotection effects to the mice. The SS2 CVCC 606 strain was used to immune the post weaning pigs and the immune sera were collected. After the purification of the antibodies from the sera, an affinity chromatographic column was prepare with the antibodies as affinity ligands used to purify the immune related antigens of the SS2 CVCC 606 strain. The antigens were analyzed by SDS-PAGE and immune to the BALB/c mice to study the effect of protection. The molecular weight of the antigens purified with the SS2 CVCC 606 strain immune sera were mainly distributed from 29.0kD to 97.2kD, which was significantly less than the whole cell antigens. There are only 60% BALB/c mice immune with the purified antigens survived when challenged with CVCC 606 strain at 5 folds of the LD50.in Conclusion ,a partly protection was obtained in mice and the immune related antigens purified in this study contained the protective compositions.In this research, The purified immune-associated antigens were identified by tandem mass spectrometry (MS/MS). Then the structure of the identified antigen were analyzed and predicted by bioinformatics. 5 important protein antigens were obtained, namely Enolase, the putative transcription regulation factor, putative ABC transporter, Predicted permease, Beta-ketoacyl synthase. Enolase was selected for prokaryotic expression and study its protection. The pGEX-4T-1 vector was choosed for prokaryotic expression, soluble recombinant protein were obtained. Western blot analysis showed that the expressed recombinant protein GST-Enolase can act with the antibodies in the serum and the protection rate on BALB/c mice was 50% when challenged with CVCC 606 strain at 5 folds of the LD50. GST-Enolase is one of important component in immune related antigen, and It could be token as an important candidate antigen for vaccine development. In this study, molecular epidemiology of Streptococcus suis were carried out in nine large farm distributing in the different area of Jilin Province, the virulence of immune-related antigens and immune function were studied with SS2 CVCC606, The antigens studied in this research could be taken as promising candidate antigens for the research of mechanisms of Streptococcus suis immune protective and vaccine development.更多还原