Studies On The Proteolytic And Chaperone Activities Of HtrA In Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae is a major pathogen of Porcine Contagious Pleuropneumonia,which causes serious economic loss to swine industry.High-temperature requirements A(HtrA)and its homologues are heat shock-induced serine proteases,which are important factors in protein regulation and important immunoprotective antigens in bacterial periplasm.In this study,we mainly carried out the preliminary study on the cleavage activity,molecular chaperone activity and immunological property of HtrA in A.pleuropneumoniae,as follows:1.The cloning and expression and bioinformatics analysis of htrA inA.pleuropneumoniaeWith the genome of APP L20 as a template,the htrA gene of 1308bp was cloned into pMD19-T vector and ligated to prokaryotic expression vector pET22b in this study.The recombinant plasmid pET22b-htrA was constructed successfully.The recombinant plasmid was identified by sequencing to show the absence of mutation,which had the nucleotide sequence consistency of 100%with L20 strain in NCBI.The recombinant expression plasmid was transfected into E.coli BL21(DE3)and expressed.With anti-His antibody as a probe,the recombinant protein was identified by Western-blotting.And the recombinant protein of about 48kDa was detected.The functional domains and epitopes of HtrA were predicted by bioinformatics methods.It was found that the HtrA included a protease domain and two PDZ domains,11 T-lymphocyte epitopes and 29 B-lymphocyte epitopes.2.The proteolytic and chaperone activities of HtrA in A.pleuropneumoniaeIn this study,the protein cleavage activity of recombinant protein rHtrA was analyzed with P-casein as a model substrate.The results showed that β-casein can be completely degraded,and rHtrA had the highest enzyme activity at 40℃ or 50℃.Using the STRING Protein Research Database to predict that the OmpP5 may be a substrate of HtrA in A.pleuropneumoniae.The recombinant protein of about 43kDa was obtained by prokaryotic expression technique.Proteolysis assays confirmed that OmpP5 was a substrate of HtrA in A.pleuropneumoniae.The total protein was extracted from normal lung tissues.The recombinant protein rHtrA and total protein of lung were incubated for 16h.The protein samples were isolated by SDS-PAGE.The results suggested that HtrA exhibited protease activity against the heat shock protein 90 in lung tissue of porcine by MALDI-TOF-TOF/MS and Western-blotting,.To determine the chaperone-like activity of rHtrA,we measured the activity of inhibiting protein aggregation with lysozyme as a substrate in vitro.The results showed that the lysozyme solution without rHtrA had obvious aggregation under the reduction of DTT,and the lysozyme solution with rHtrA had no obvious aggregation,which confirmed that rHtrA showed chaperone activity to lysozyme.3.The research of the immune characteristics of HtrA in A.pleuropneumoniaeRecombinant protein rHtrA(50μg),inactivated strain L20 was mixed with Gel01 aqueous adjuvant thotoughly.Multi-point injection by subcutaneous to immunize each mouse.And the control group of adjuvant and PBS was setted up in this study.The second immunization was carried out after]4 days.Serum samples were collected by cutting off tails in the 7th day after the second immunization.The result showed that recombinant protein rHtrA could induce the specific antibody titer of 1:25600 in mice.Further to detect subtype of IgG,group of rHtrA induced IgG2a-based antibody.At the same time,the cytokines in the serum of the immunized mice were IL-2 and IFN-γ predominantly,which indicated that HtrA mainly induced mice to produce Th1 cells.The result of lymphocyte proliferation showed that recombinant protein rHtrA can promote a significant proliferation of spleen lymphocytes in vitro.The mice were challenged with intraperitoneal injection of APP L20 at 2.3×108CFU(5×LD50)and the morbidity and mortality were counted.The results showed that all the mice died in control group,and survival rates of the groups of rHtrA and L20 were 37.5%and 87.5%,respectively.These studies indicated that HtrA is a immunoprotective antigen in A.pleuropneumoniae.