Study On Cloning, Expression And Immunity Of The Hsp65 Gene Of Mycobacterium Avium Subsp. Paratuberculosis

Paratuberculosis disease caused by Mycobacterium avium subsp.Paratuberculosis (MAP,Mycobacterium paratuberculosis) induce a series of characteristics as chronic hyperplastic enteritis andprogressing extenuation in ruminants also called Johne's disease.It causes the cut down of foragereward,milk secretion and productivity in animals and result in great loss to animal husbandry.Thisdisease has received extensive attention globally for its potential relation to human Crohn's disease.Atpresent,there is no effective drug to treat this disease and for eradicating source of infection andreinforce breeding management are the major preventive measures including allergy,ELISA,separationand identification of pathogen.Then isolate or reject the positive cattle.In addition,vaccination withinactivated or attenuated MAP also can be used to control the disease.Although the cut down of milkproduction can't be prevented by immunization but the clinical cases can be reduced 90% and theamount of sub clinical infectious animals and positive cases detected by histology or bacteriology alsowere reduced.But that immunized with whole strain vaccine will interfer the allergy and serologicdetection so this method has been forbidden in many countries.Therefore the exploitation of newgeneration vaccine for paratuberculosis disease is imperative.Heat shock proteins (HSPs) are highly conservative protein synthetized by organism whenstimulated by physical,chemical,biological and mental factors.Main HSPs can be divided into 5families on account of molecular weight including macromolecule weight family,HSP90 family,HSP70 family,HSP60 family and micromolecule weight family.HSP60 as a kind of molecularchaperones is a kind of immunodominant antigen in the infectious process of many pathogens and it canassist recovery of native conformation of degenerated and indissoluble agglomeration protein.Hsp65gene also called groEL2 gene and its codogenic protein is HSP60 family.This protein not only is themajor protective antigen but also has function of submitting antigen assisting dendritic cell.Hspl0(GroES) as a kind of assisting molecular chaperones can assist the protein folding directed byGroLS.As a kind of efficient T cell antigen,it can stimulate the organism generate specific cellullarimmunologic response and induce the generation of IFN-γby proliferation of peripheral bloodmonouclear cells in PPD dermal test positive cases.It also can be used as alternative antigen of newgeneration vaccine.In this experiment,we screen the major protective antigen hsp65 gene of MAP in order to developnew vaccine especially the DNA vaccine for the prevention of paratuberculosis disease.The hsp65 genewas amplified from MAP C-2 chromosomal DNA by using the PCR technique.We gained a hsp65 gene of 1 626bp.Then PCR product was cloned into pGEM-T vector by T-A clone technique and therecombinant clone was identified by plasmid size,enzyme digestion and PCR identification.The cloneplasmid of pGEM-T- hsp65 was successfully constructed.The nucleotide sequence and deduced aminoacid sequence ofclone gene was analyzed by DNASTAR software.The result indicated that the size ofhsp65 gene consist with M.paratuberculosis K-10 strain in GenBank and the sequential homogeneityreached 99.1%,the amino acid homogeneity reached 99.3%.The preceding analysis indicated that thehsp65 gene was very conservative in M.paratuberculosis.The hsp65 gene that was digested withenzyme from pGEM-T- hsp65,purified and then subcloned into a pET-32a(+) procaryotic expressionsystem,then the procaryotic expression plasmid pET-32a- hsp65 was constructed successfully.Theplasmid was transformed into E.coli BL21(DE3) and induced by IPTG.We gained a 76Ku fusionprotein.The expression protein was analyzed using Western-blot,which proved that it had the antigenicactivity of Mycobacterium avium subsp.Paratuberculosis.Base on above study,in order to study DNA vaccine of hsp65 gene the major protective gene ofMAP.Hsp65 gene from recombinant plasmid pGEM-T-hsp65 was subcloned into the eukaryoticexpression vector pVAX1,then the eukaryotic expression plasmids pVAX1-hsp65 were successfullyconstructed.Transfect BHK-21 cells with the constructed recombinant plasmid pVAXI-hsp65 bycationic polymerization gene transfection reagent and observe the expression in mammalian cells ofhsp65 gene by immunofluorescence and RT-PCR.Meanwhile hsp65 gene from recombinant plasmidpGEM-T- hsp65 was subcloned into pVAX1-hsp10 and the eukaryotic expression recombinant plasmidpVAX1- hsp65-10 was constructed successfully.BALB/c mice was vaccinated with pVAX1-hsp65 and pVAX1-hsp65-10 recombinant plasmid,moreover,pVAX1 and sodium chloride as control.Detection of specific antibody shows that and thereis a generation of specific antibody against hsp65 and the antibody titer in bivalent gene group is higherthan single gene group.Lymphocyte transformation test show that cell proliferation activity in pVAX1-hsp65 and pVAX1- hsp65-10 group was higher than sodium chloride control group and there is nosignificant difference between pVAX1- hsp65 and pVAX1- hsp65-10 group (P＞0.05).CD3~+T,CD4~+T and CD8~+T ceils determination results demonstrated that CD8~+T cells in pVAX1-hsp65 andpVAX1- hsp65-10 group were more than the control group with non-significant difference (P＞0.05 ).This also shows a MHCⅠtype cell immune response in experimental group.The amount.of CD4~+Tand CD3~+T cells in pVAX1-hsp65 group is significantly higher than the other groups.The IFN-γlevelin pVAX1- hsp65 and pVAX1- hsp65-10 group were higher than control group,but not yet reached astatistically significance (P＞0.05).However the result show that recombinant plasmids immunizationstrengthened the cell immunity of mice,and bivalent gene group is higher than single gene group. Cavia cobaya was vaccinated with pVAX1- hsp65 and pVAX1- hsp65-10 recombinant plasmid,moreover,pVAX 1 and supersonic antigen of MAP as control.The allergy quarantine with avian bacillustuberculosis PPD indicated that there is significantly difference between the pVAXl-hsp65,pVAX1-hsp65-10 groups and supersonic antigen of MAP group (P＜0.01).This indicates that the Hsp65 andHsp 10 does not interfere with detection of Paratuberculosis allergy.