Screening Of The Mycoplasma Hyopneumoniae Vaccine Candidate Proteins

Mycoplasmal pneumonia of swine(MPS)caused by Mycoplasma hyopneumoniae(Mhp)is a chronic,debilitating and transmitted respiratory system disease,based on the slow cough and asthma as the main symptom which causes considerable economic losses in the swine industry.At present,the inactivated vaccine is applied to prevent this disease.Whereas,attenuated vaccine is also used,though the popularization and application is limited.There are still some problems for these commercial vaccines,such as the poor effect,high price and inconvenience.Therefore,it is very important to develop more effective and convenient vaccines against MPS.1.The establishment of the ELISA method for identification of serological humoral immunodominant proteins of Mycoplasma hyopneumoniaeMhp366 is a Mhp protein that can stimulates the host to produce strong humoral immunity in natural infection.It can react with convalescent serum and did not react with serum from inactivated bacterin-immunized pigs.After constructing the recombinant plasmid p GEX-6P-1-mhp366 and transforming it into Escherichia coli BL21(DE3),the GST-Mhp366 protein was induced and expressed by the recombinant bacteria E.coli GST-Mhp366 successfully.The lysates of E.coli GST-Mhp366 and engineering bacteria E.coli GST were added into the glutathione coated plates and reacted with 11 convalescent sera and 7 negative sera,respectively.Then,the optimization of experimental conditions,including the coated antigen,blocking buffer,and dilutions of sera and second antibody,were determined.The optimal concentration of the coated antigen was the original bacteria lysates without dilution,and the optimal blocking buffer contained 10% FBS and 2.5% skim milk in PBS.Besides,the working concentration of serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were 1:500 and 1:40 000,respectively.Thus,an indirect ELISA was established for identification of serological humoral immunodominant proteins of Mhp.2.Screening and identification of some serological humoral immunodominant proteins of Mycoplasma hyopneumoniaeTweenty conserved universal existed Mhp proteins were screened by using bioinformatics method.These proteins are Mhp153,Mhp156,Mhp228,Mhp252,Mhp265,Mhp322,Mhp336,Mhp351,Mhp364,Mhp379,Mhp390,Mhp424,Mhp472,Mhp483,Mhp504,Mhp511,Mhp535,Mhp623,Mhp677 and Mhp682.The genes of the 20 proteins were ligated into p GEX-6P-1,then transformed into E.coli BL21(DE3)or E.coli XL-1 Blue and induced to express the recombinant proteins successfully.The lysates of 20 E.coli recombinant bacteria and engineering bacteria E.coli GST were added into the glutathione coated plates and reacted with 11 convalescent sera and 7 negative sera,respectively.Ten percent FBS and 2.5% skim milk in PBS was used as blocking buffer,and the serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were diluted by 500 times and 40 000 times for the ELISA assay,respectively.Finally,10 serological humoral immunodominant proteins of Mhp were identified,namely Mhp153,Mhp156,Mhp336,Mhp364,Mhp379,Mhp424,Mhp472,Mhp535,Mhp623 and Mhp677.3.The establishment of the ELISA method for identification of serological humoral immunodominant proteins of Mhp that can distinguish convalescent sera and inactivated bacterin-induced hyperimmune seraThe glutathione-coated plates coated with original lysates of E.coli GST-Mhp366 and engineering bacteria E.coli GST without dilution and blocked with PBS adding 10% FBS and 2.5% skim milk were reacted with 11 convalescent sera and 7 inactivated bacterin-induced hyperimmune sera,respectively.The optimization of experimental conditions,including dilutions of sera and second antibody were determined.The working concentration of serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were 1:500 and 1:20 000,respectively.Thus,an indirect ELISA was established for identification of serological humoral immunodominant proteins of Mhp that can distinguish convalescent sera and inactivated bacterin-induced hyperimmune sera.4.Screening and identification of some serological humoral immunodominant proteins of Mycoplasma hyopneumoniae that can distinguish convalescent sera and inactivated bacterin-induced hyperimmune seraThe lysates of the 10 E.coli recombinant bacteria whose recombinant proteins were identified as serological humoral immunodominant proteins and engineering bacteria E.coli GST were added into the glutathione coated plates and reacted with 11 convalescent sera and 7 inactivated bacterin-induced hyperimmune sera,respectively.Then,10% FBS and 2.5% skim milk in PBS was used as blocking buffer,the serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were diluted by 500 times and 20 000 times,respectively.Finally,Mhp336 was identified as the serological humoral immunodominant protein of Mhp that can distinguish convalescent sera and inactivated bacterin-induced hyperimmune sera.It can provide the target antigen for the development of the genetic engineering subunit vaccine against MPS.