Study On The Components Of Membrane Protein And Its Immunogenicity In M.mycoides Subsp.capri

Contagious caprine pleuropneumonia(CCPP)was a acute or chronic contagious disease that occur specially in goat.There are always pathological changes in the thoracic cavity of caprine infected with CCPP.clinically it mainly present ardent fever,cough,respiratory passage rales, catarrhus blenna narium,conjunctivitis in eyes,fibrinous pleurisy,pneumonia,and some female elder abort,constantly emaciate.CCPP is considered contagious disease in B category. M.capricolum subsp.Capripneumoniae(Mccp)was for many years designated as the classic causative agent of CCPP,which was designated as biotype strain F38 before,strain F38 belongs to the so-called "Mycoplasma mycoides cluster," which includes the following mycoplasmas.M.capricolum subsp. Capripneumoniae(Mccp),Mycoplasma capricolum subsp,capricolum(Mcc),Mycoplasma mycoides subsp.Mycoides SC(MmmSC),M.mycoides.subsp mycoides Large colony LC(MmmLC); M.mycoides subsp.capri,(Mmc);Mycoplasma subsp,bovine group 7,(Mbg7).Reporting so far,the etiological agents of CCPP domestic mainly include Mccp,Mmc,Mycoplasma ovipneumoniae(MO), MmmSC.This study to aim at identify the etiological agent of CCPP with molecular biology method, which isolated from ChongQing,and preliminary study on the components of membrane proteins and its immunogenicity.Inorder to construct effective molecular biology method to the epidemiological investigation of CCPP in ChongQing and to supply the rationale for the preparation of subunit vaccine or diagnostic antigen.Two primer were design respectively according to the highly conserved sequence 16S rRNA of mycoplasma and lipoprotein sequence LppA of MmmLC and Mmc。We used this two primer to identify the etiological agent of CCPP which isolated from ChongQing.DNA was prepared by saturate Tris-phenol,PCR was run by optimize reaction system.PCR turn out that objective strap were amplified in both primer,while none in control group.This PCR reaction system can detecte lower limit 10ng / mL DNA.According to the result of PCR the etiological agents of CCPP which isolated from ChongQing were identified as Mmc.Preliminary study on the components of membrane proteins and its immunogenicity to make clear some membrane protein fragment which possess immunogenicity and then find out protective antigen fragment,providing theory foundation to prevent CCPP.Thallus were quassation by supersonic wave and then SLS was used to dissolve membrane protein,at last using ultracentrifugation to precitipate membrane protein.The components of membrane proteins was analyzed by SDS-PAGE,staining with Coomassie Blue R-250 and silver recpectively.It turned out that molecular weight of membrane protein of PG3 is between 99.7kDa-15.4 kDa,altogether 11 fragments,molecular weight is recpectively 99.7kDa,81.9 kDa,75.1 kDa,65.6 kDa,57.4kDa, 51.1kDa,27.8kDa,25.2 kDa,20.1 kDa,16.8 kDa,15.4kDa.And that of field strains are most consistent,their molecular weight of membrane protein is between 100.6 kDa-17.1 kDa,altogether 13 fragments,molecular weight is recpectively 100.6 kDa,94.5 kDa,81.3 kDa,75.4 kDa,72.7 kDa, 65.1 kDa,62.4 kDa,51.3 kDa,48.0 kDa,33.2 kDa,25.1 kDa,20.1 kDa,17.1 kDa.There are not obvious difference in the membrane protein between the field strains,and that of filed strains are alike,there are 4 same fragments between field strain and type strain:81.9 kDa,75.1 kDa,65.6 kDa and 51.1kDa.The membrane protein was quantitated and emulsionized with adjuvant and then used to immune rabbit in order to get antiserum.Bleeding and separating serum when the potency achieved 1:16,IgG was purified by saturated ammonium sulfate,which was used to immunoblotting reaction.At the same time,bleeding and separating serum fortnightly after immunity,indirect ELISA was used to detect the valence of antibody in 7 weeks,and draw the curve.It turned out that all the strains produced specific antibody in one week after immunity,valence of antibody was 1:400-1:800. The valence of antibody ascend the peak value 1:6400-1:12800.And the valence of antibody maintain at 1:3200-1:6400,while the valence of antibody descend hurry up after immunity in 6 weeks.Which showed that the valence of antibody was proportional to the immunity periods and immunity times.Take blood fortnightly after first immunity,equal quantity lymphocyte separated medium was added to anticoagulated blood(with heparin sodium anticoagulation).Lymphocyte conversion ratio(LTR)was detected by methyl thiazolyl tetrazolium(MTT)colerimetry.Membrane protein was added to experimental group make it final concentration 500μg/mL,and control group was added equal amount DMEM with 10%fetal calf serum(FCS),MTT was added before cultivation terminated and 50%DMF-20%SDS was added after cultivation terminated,shaking for several second and standed in dark for one hour.And then fech OD₅₆₀,LTR was calculated according formula.Results show that all of membrane protein can stimulate lympholeukocyte multiplication in vitro obviously,which indicate that experimental rabbit have specific immune response to membrane protein,thus it was certified that there was strong immunogenicity in these membrane protein.Purified IgG and membrane protein were used to immunoblot.Eliminating nonspecific reaction was perform by membrane protein and negative serum.Every membrane protein first reacted with, with the view of discovering some fragments that can stimulate organism generate antibody.And then membrane protein and antiserum of type strain and field strains cross reacted with each other in order to verify that there were whether or not some common protective antigen fragments between the two.Results show that when PG3 reacted with its homologous there were 4 strap,respectively 94.5 kDa,65.6 kDa,62.5 kDa,53.0 kDa.And there were 4 strap that of field strains respectively 101.4 kDa,84.9 kDa,62.5 kDa,53.0 kDa.When PG3 antiserum reacted with filed strain,cross reaction was seen in 65.6 kDa and 53.0 kDa,the same result was seen when membrane protein of PG3 reacted with antiserum of field strains.Which indicated that there were some common protective antigen fragments between the two,and they have common protective antigen fragments between field strains.