A Study Of Coxsackievirus A16 VP1 Subunit Vaccine

Coxsackievirus A16(CVA16)belongs to the genus Enterovirus of the picornavirus family and is one of the major pathogens of hand foot and mouth disease(HFMD).In recent years,it has been found that CVA16-infected patients can cause secondary infections such as brain,lung,and heart,and may even cause fatal complications such as pneumonia,myocarditis,and refractory shock.E.coli heat-labile enterotoxin(LT)is a bacterial toxin and has mucosal immune adjuvant activity.LT can induce mucosal immune responses and regulate the generation of cytokines and the formation of T cells by increasing the permeability of epithelial cells and regulating the differentiation of B cells..Escherichia coli heat-labile enterotoxin B subunit(LTB)can be used as a mucosal adjuvant for specific antigens to enhance serum IgG antibody responses against specific antigens.The Escherichia coli type II heat-labile enterotoxin B subunit(LT2B)has stronger adjuvant activity than LTB.Objective: The study of the immunogenicity of CVA16VP1 and LT2 B fusion proteins as antigens,and preliminary evaluation of antibody titers produced by the antibodies,will lay the foundation for further research on novel vaccines against Coxsackie virus.Methods: In this experiment,the synthesized CVA16VP1 was amplified by DNA polymerase,a large number of CVA16VP1 gene fragments were obtained,and two restriction sites of BamH I and EcoR I were introduced,and were digested and then ligated into the pET32a(+)E.coli expression vector.The pET32-CVA16VP1 expression plasmid was transformed into E.coli BL21(DE3)competent cells.A large number of LT2 B gene fragments were amplified by designing primers against LTB,and the restriction sites of Sal I and Not I were introduced into the plasmid.After restriction enzyme digestion,the fragment was inserted into pET32-CVA16VP1 to construct pET32-CVA16VP1-LT2 BO expression vector and transferred into BL21 competent cells.Designed primers for full-plasmid amplification of pET32-CVA16VP1-LT2 BO,introduced another EcoR I restriction site,EcoR I single cut PCR product,self-ligated to obtain pET32-CVA16VP1-LT2 B expression vector,and transferred to BL21 competent cells.Through plasmid PCR detection,enzyme digestion assay,and sequencing assay,we determined the correctness of the constructed vector.On the basis of previous research in the laboratory,the optimal conditions for inducing protein expression were determined.The correctness of the target protein expression was detected by Western-blot.The protein was purified by using a His-tag magnetic bead.TGE dialyzed protein renaturation.Polyethylene glycol 8000(PGE8000)was used to concentrate the purified low-concentration protein to obtain a high-purity concentration of CVA16VP1-LT2 B fusion protein.CVA16VP1-LT2 B protein was used to immunize Balb/c mice by intranasal immunization.Serum of mice was collected and specific IgG levels in serum were detected by indirect ELISA.Results:1.The pET32-CVA16VP1-LT2 B fusion expression vector was successfully constructed and successfully transferred into BL21 competent cells.2.The CVA16VP1-LT2 B fusion protein with his-tag was expressed in BL21,and the target band was detected by Western-blot,which was about 66 KD.3.Mouse sera were obtained by intranasally immunizing mice three times,and specific antibodies were produced in the serum of the mice by Western-blot assay.4.The results of specific antibody titer in mouse serum by indirect ELISA detection showed that CVA16VP1-LT2 B has a certain degree of immunogenicity and the antibody titer produced is 1:800.Conclusion: The successfully constructed pET32-CVA16VP1-LT2 B fusion expression vector can express an immunogenic fusion protein,and this fusion protein is our target protein.By vaccinating mice three times for a total of 28 days,sufficient serum can be obtained and specific antibodies can be successfully expressed.The antibody titer of this antibody can be detected by indirect ELISA at 1:800.