The Expression Of The Main Antigen Of Mycoplasma Hyopneumoniae In Pichia Pastoris And Establishment Of An Indirect ELISA For Detection Of Antibody

Mycoplasma hyopneumoniae (Mhp) is the causative agent of Swine Enzootic Pneumoniae, a disease which is characterized by highly infective,chronic,high morbidity and low self-mortality.Infected swine has a retarded growth and inefficient food conversion.Mhp destroy the respiratory tract mucosa-cilia barrier.so it can induce the infection of other pathogenic bacteria and increase the mortality, this disease exits worldwide and cause a severely economic losses. Currently there have no better sero detecting method in our country,and at present most research focus in the expression of antigenal protein in the E.coli,then purified it to establish ELISA detection method. But the fused protein and the constituent of E.coli may interfere the application of the expressed protein. In this experiment,we expressed main antigenal protein of Mycoplasma hyopneumoniae in pichia pastoris and developed indrect ELISA detection method for Mhp antibody.The study was unfolded through the following aspects.1 Accroding to the sequence of P36 gene of Mycoplasma hypneumoniae from GenBank (GenBank Mhp AE017243),a pair of primer was designed to amplify the whole P36 gene.Then the gene was cloned into the expression vector pPICZa-A to construct the recombination expression vector pPICZa-A-P36.The recombinant vector was linearized by Sac I and was electransformed into P. pastoris X-33 strain.Under the control of the promoter AOX1 (alcohol oxidase 1)and inducing with methanol.SDS-PAGE and Western-blotting analysis showed that the gene could be expressed in pichia pastoris and the product has good reacting ability.2 We studied the relation between expression yield and growth conditions and found the optimal expression conditions of the recombinant P36 protein were:28-30℃,200rpm,the recombinant bacteria were incubated in BMGY for 24h,then transferred it into new BMMY medium of pH5.5 which with OD600 being 1.5,then the solution was incubated for 72h and methanol was added to keep its concentration up to 1.0% per 24h.After induction under optimal conditions,the yield of P36 protein could be added to 285μg/mL.The purified protein as coating antigen was established to monitor antibodies level of pigs against Mhp P36 and the operation method was optimized.The optimal operation methods were:The protein concentration of 35μg/mL was coated onernight at 4℃on 96-well microtiter plates and the plates were subsequently blocked 3h with 1% gelatine.Then incubated for 60min with sera diluted to 1:80 by PBS,the working concentration of HRP-goat anti-swine IgG was l:30000,the chromogenic time was incubated at 37℃for 15min.3 Accroding to the sequence of P97 gene of Mycoplasma hyopneumoniae from GenBank(GenBank Mhp AE017243), a pair of primer was designed to amplify the P97R1 gene.Then the gene was cloned into the expression vector pPICZα-A to construct the recombination expression vector pPICZa-A-P97R1.The recombinant vector was linearized by Sac I and was electransformed into P. pastoris GS115 strain.Under the control of the promoter AOX1(alcohol oxidase 1)and inducing with methanol.SDS-PAGE analysis showed that P97R1 protein could be highly expressed in pichia pastoris and Western-blotting analysis showed that the product has good reacting ability.4 An indirect ELISA using the purified protein P97R1 as coating antigen was established to monitor antibodies level of pigs against Mhp P97R1 and the operation method was optimized.The optimal operation methods were:The protein concentration of 31μg/mL was coated onernight at 4℃on 96-well microtiter plates and the plates were subsequently blocked 3h with 5% skimmed milk.Then incubated for 60min with sera diluted to 1:20 by PBS,the working concentration of HRP-goat anti-swine IgG was 1:10000,the chromogenic time was incubated at 37℃for 15min.