The Research Of Typing Haemophilus Parasuis And Cloning Expression Of TbpA Gene And Analysis Of The Protection Of The Expressed Protein

Haemophilus parasuis is the etiological agent of Glasser’s disease in pigs, whose mainsymptoms are fibrinous polyserositis, polyarthritis and meningitis; furthermore, somestrains can also be found as a commensal of the upper respiratory tract in healthy pigs.With the recent changes in production methods, the disease has become one of the mostimportant bacterial diseases in the pig-rearing industry worldwide. To date,15serovarshave been recognized with most recent serotyping studies noting the existence ofnon-typable isolates, indicating the possibility of further serovars. Though some studieswere designed to develop killed whole cells or live avirulent vaccines, the existence ofdiverse genetic make-ups made the results ambiguously. Due to lack of thoroughknowledge of the virulence factors and protective antigens, novel vaccine development hasbeen hindered to against the disease and the epidemiological study.The study reported here focused on the serovar distribution in Anhui province andcloning, expression the tbpA gene of Haemophilus parasuis to research theimmunogenicity and protective in mice.There are two main purpose:(1) Understand HPSepidemic in Anhui serotypes, ERIC-PCR and PCR-RFLP polymorphism, so as to exploreareas of Anhui HPS epidemiology and molecular epidemiology of serum lay thefoundation for effective Glasser’s disease prevention and control provide a scientific basis;(2) Sub cloning and expression of HPS tbpA discussed the role of sub-protective immunityproteins for the prevention and control and the development of new HPS subunit vaccinebasis.1Serological characterization and ERIC-PCR and PCR-RFLP genotyping of Haemophilusparasuis isolates from Anhui ProvinceFrom different regions in Anhui,18strains of HPS were isolated from2006-2010,based on morphologic, cultural and bolchemical characteristics. All HPS isolates wereserotyped by KRG gel diffusion test and ERIC-PCR, PCR-RFLP anlysis.Three HPS serovars were indentified. Serovar4（27.78%,5/18） was the most prevalentserovars, followed by serovars14（16.67%,3/18）and serovars13（11.11%,2/18）, while44.4%of the isolates could not be assigned to a serovar(nontyable).18HPS isolates weregenotyped with the enterobacterial repetitive intergeneic consensus based-polymerasechain reaction (ERIC-PCR) technique and11distinct strains were identified. Genotypes IV（27.78%,5/18） was the most prevalent genotypes, followed by genotypes II （16.67%,3/18）and genotypes I（11.11%,2/18）.18HPS isolates were analysed by usingPCR-RFLP based on tbpA gene produced6different patterns. And the first3genotypes ofmost prevalence in Anhui province were DBP（44.44%,8/18）,BAE（22.22%,4/18）and BBB（16.67%,3/18）.This result also showed polymorphism. The strains which can not bedivided were also obtained Effective typing. The results showed that serotypes, ERIC-PCRgenotype, PCR-RFLP genotype had no significant correlation between the three, while thesame ERIC-PCR genotypes and sources of related serotypes.2Cloning and expression the tbpA gene of HPS and research the protective efficacy inmiceThree pairs of specific primers were designed according to the tbpA gene sequence ofHPS SH0165strain from GenBank number7277767, three fragments of tbpA1, tbpA2andtbpA3containing poly-antigen epitopes were amplified and were cloned into pET-32a(+)vector respectively. It could be found that all the three fragments were expressed with thesize of62kD、54kD、44kD by SDS-PAGE.The results of western blotting analysis demonstrated that three fusion proteinpossesed antigenic activity could be specifically recongnized by antiserum against HPS.The mice were vaccinated subcutaneously in0day,10days and20days. The antiserumwas taken before the day of the first vaccine,10days and20days. And the antibody isdeterminded by ELISA. After7days of the third vaccine, the mice were challenged with5×LD50dose of HPS LJ3strain. The survival rate of the TbpA1and inactivate bacterin is20%and40%. Bctericidal assays showed that the TbpA1of rabbit polyclonal antibodiescould against the HPS LJ3strain. The results indicate that rTbpA1can partly protect micefrom the challenge of HPS LJ3strain.