Cloning And Immunogenicity Of Haemophilus Parasuis LolA And ThyA Proteins

Haemophilus parasuis(HPS)is one of the current bacterial diseases that mainly affect the development of intensive pig industry.HPS can cause multiple organ lesions in sick pigs,and is easy to be mixed with a variety of bacterial and viral diseases,greatly increasing the mortality of sick pigs.The cause of Haemophilus parasuis is severely resistant,and it is difficult to rely on drugs to control its spread and occurrence.In addition,existing commercial vaccines have low cross-immunogenicity due to different bacterial antigens,and no vaccine can be used for all.The serotypes all produce effective immune protection.Therefore,in order to control the occurrence and prevalence of HPS,some researchers are developing subunit vaccines to prevent HPS.The immunological activity of HPS LolA and thyA recombinant proteins was identified.The expression of LolA and thyA secreted proteins of HPS was obtained and purified by prokaryotic expression system.The immune effect of recombinant protein was identified by using Kunming mouse model.The results showed that:(1)by comparing BLAST with all 10 strains of HPS strains in Gene Bank,it was found that the LolA and thyA genes were conserved in the HPS strain;(2)the purified recombinant proteins LolA and thyA were harvested and passed the His-tagged antibody.Western-blot screening with 13 HPS-positive sera obtained fragments with the same size;(3)The detection of antibody Ig G showed that the inactivated group was significantly higher than the negative control group(p<0.05),while the recombinant protein LolA The recombinant protein thyA and the mixed protein group Ig G levels were significantly higher than the negative control group(p<0.01);indicating that the recombinant protein LolA,thyA and the mixed protein group can stimulate the production of specific antibody Ig G in mice,and excellent In the whole bacteria inactivated group.(4)Compared with the blank group and the negative control group,IL-4 cytokine levels were significantly increased(p<0.01),IFN-? and IL-10 levels were significantly increased(p<0.05);Cytokines IFN-? and IL-4 were significantly higher than the blank group and the negative control group(p<0.01),and IL-10 was also significantly increased(p<0.05).(5)The levels of recombinant protein thyA IL-4 and IL-10 were significantly higher than those in the blank group and the negative control group(p<0.05),and higher than the whole bacterial inactivation group.Therefore,the recombinant protein LolA,mixed protein LolA,thyA can stimulate the body to produce Th1 and Th2 immune responses to a certain extent,while the recombinant protein thyA and whole bacteria inactivated group only have a certain effect on Th2 immune response.(6)The recombinant protein LolA can provide 50% protection against lethal dose of type 13 HPS.The mixed proteins LolA and thyA can only provide 40% protection rate,indicating that recombinant protein LolA,mixed protein LolA,thyA pair 13 HPS has a certain protective capacity and can be used as a new vaccine candidate antigen,laying a foundation for the development of a safe and efficient new subunit vaccine of Haemophilus parasuis.