Construction Of HSV-2Epitope Recombinant Mycobacterium Smegmatis And Immune Effect

ObjectiveConstruction of herpes simplex virus type2vector vaccine, immune effect ofdetection of recombinant vector vaccine, for the prevention and treatment of lay atheoretical foundation for recombinant vector vaccine of herpes simplex virus type2infection, provide a new strategy for the development of herpes simplex virus type2vaccine. Perfect the construction technology of Mycobacterium smegmatis vaccine, for itas virus vaccine preparation platform to provide technical support.Methods1）Construction of the plasmid pmv261-gBD-570Literature, according to the sequence of HSV-2gD gene and gB GeneBank registrationin the construction of gBD-570, containing EcorI and salI restriction sites and subclonedinto puc57gBD-570, was identified to be correct, at the same time restriction shuttleplasmid PMV-261and puc57-gBD-570, and the pmv261plasmid gBD-570fragments weresubcloned into the recovery after digested in construction of recombinant plasmid,PMV261-gBD-570, the plasmid, restriction and colony PCR after correct identification ofrecombinant plasmid,-20℃preservation reserve.2）Expression of gBD-570gene proteinThe extracted plasmids by electroporation was subcloned into the M.smegmatis(MC2155), with the expression of SDS-PAGE method for identification of gBD-570.3) immune animalFemale BALB/c mice were randomly divided into6weeks of age into6groups (n=10), in the mouse femoral four muscle injection, each group is as follows: group1, andeach side of the injection of NS (normal saline)200μ L; group2, each side injection is empty vector PMV261200μ L (1mg/ml); group3, each side with1×106cfu/ml200μMC2155L (1mg/ml); the4group, injection of low concentration (1×104cfu/ml) of therecombinant MC2155200μ L; group5, injection concentration (1×106cfu/ml)recombinant group MC2155200μ L; group6, high concentration (1×108cfu/ml)recombinant group MC2155200μ L. Three weeks after immunization, mice tail blood,serum IgG, the expression of IFN-γ and IL-2indirect ELISA method, and through theMTT method on the mice spleen lymphocyte proliferation rate were detected.Results1）The recombinant plasmid PMV-gDBD570was transformed into E. coli DH5α,through colony PCR, double enzyme digestion and sequencing, the recombinant plasmidswere successfully constructed.2）The constructed plasmid electroporation successfully transformed intoM.smegmatis MS, through colony PCR and enzyme digestion identification, plasmids.3）The results of SDS-PAGE showed, the recombinant plasmid could be expressedcorrectly in MS.4）The experimental result shows: after immunization of mice with immune effect ofrecombinant MC2155concentration group and high concentration group MC2155werebetter than those of the other experimental group, can induce humoral and cellularimmunity.Conclusion1）Successfully constructed fusion protein expression vector PMV261-gBD-5702）Effective expression of recombinant expression vector in M.smegmatis3) Recombinant Mycobacterium smegmatis can induce humoral and cellularimmunity of mice, and laid the foundation for the next vaccine preparation work...