Expression Of CVB3 VP1 Protein In Prokaryotic System And Observation Of Its Immune Effect

Objective: Coxsackievirus group B (CVB) is a leading pathogen of human viral myocarditis. Among the six serotypes of CVB, CVB3 is the most important. The VP1 is a major capsid protein of CVB3, which contains many antigenic epitopes of B and T cells and can induce effective immune responses in mice. It was shown that the plaxmid-based DNA vaccine of VP1 could induce humoral and cell mediated immune responses in mice, but the responses were usually too weak to protect the host from lethal challenge of CVB3. The reason was supposed to be that the vaccine DNA entered into the cells was too few or the antigen encoded by the DNA expressed poorly. Protein has been proved to be the strongest immunogen that can elicit the immunized animal to produce higher level production of antibody. Considering the reasons above, people are trying again to use protein or peptide in the development of vaccine, expected to get more effective vaccines. The proteins or peptides used are extracted from the pathogens or obtained by gene engineering, which are component of the pathogen and so called subunit vaccines. These vaccines are noninfectious, not pathogenic and so do not need inactivating. Since the VP3 gene or foot and mouth disease virus was cloned into E.coli and the protein produced was used as vaccine in 1981, many subunit vaccines have be developed successfully, which include HBV subunit vaccine. But on the other hand, some investigations showed that the immunogenecity of some subunit vaccines were too weak to provide the host effective immune protection. The prime/boost strategy by combined immunizing the animals with DNA and subunit vaccines has been shown to be more effective recently. In this study, a subunit vaccine, CVB3 VP1 was prepared from prokaryotic system and evaluated with a CVB3 VP1 DNA vaccine, pCDNA3/VP1, for their immune effect in different prime/boost combinations.Methods: 1 CVB3 VP1 gene was amplified by PCR and cloned into pGEM-T vector. The E.coli DH5αwas then transformed and cultured in medium containing Ampicilin. The plasmids were extracted and identified by restriction endonuclease digestion as well as sequencing. 2 VP1 fragment was obtained from pGME-T/VP1 by proper endonucleases digestion and then inserted into pET-His vector that was cut by the same endonucleases to construct a prokaryotic expressing plasmid of pET-His/VP1. 3 The expressing bacteria BL21 (DE3) pLysS was transformed with pET-His/VP1 and VP1 fused with a histidine X 6 tag was induced by IPTG. The protein produced was verified by Western blot analysis using the Histidine and CVB3 antibodies. The transformed bacteria were broken by ultrasonic treatment and then the purified inclusion was obtained. 4 Large preparations of plasmids of pcDNA3/VP1 were performed and then purified by PEG8000. 5 BALB/c mice aged 6-8 weeks were divided into five groups: PBS group, plasmid group, protein group, plasmid/protein group, protein/plasmid group, with eighteen mice in each. The mice were immunized three times at three weeks'interval with 100μg of plasmid or protein respectively. The plasmid was diluted by PBS and injected in quadriceps. Diluted by PBS, the protein was given intraperitoneally. Complete Freund's adjuvant was used in the first inoculation of protein, and then incomplete Freund's adjuvant. Fourteen days after every injection, sera were collected and tittered for CVB3-specific neutralizing antibodies; three weeks after the third immunization, splenocytes from three mice of each immunized group were stimulated by inactivated CVB3 and the lymphocytic proliferative activity and specific CTL cytotoxic activity were tested by CCK-8 assay; In the same time, the other twelve mice were challenged with 4LD50 CVB3 and the amount of surviving animals was monitored up to three weeks post infection; Furthermore, the rest mice of each group were challenged with 3LD50 CVB3 and sacrificed seven days later in order to evaluate the virus titers in blood.Results: 1 CVB3 VP1 fragment was amplified by PCR. DNA sequencing showed that VP1 was identical to the sequence recorded in Genbank. 2 The prokaryotic expressing plasmid of pET-His/VP1 was constructed successfully. 3 VP1 protein was expressed in prokaryotic expression system successfully. Expressed as inclusion form, the protein was verified by Western blot analysis against the His and CVB3 antibodies. 4 The mean titers of neutralizing antibody after every immunization in plasmid group were 1:5.62, 1:15.85, 1:23.71, and that in protein group were 1:8.91, 1:44.67, 1:70.79, and that in plasmid/protein group were 1:5.62, 1:15.85, 1:33.5, and that in protein/plasmid group were 1:8.91, 1:44.67, 1:56.23, and that was always lower than 1:5 in PBS group. The statistics analysis showed that excepting PBS group, the differences of neutralizing antibody titers of every group were significant after every times of immunization. More specifically, mice in protein group, plasmid/protein group and protein/plasmid group elicited higher titers of neutralizing antibody after the third immunization. When compared with that of plasmid group, the difference was significant (P<0.05). 5 Splenocytes from the vaccinated mice showed different proliferative activity when stimulated by CVB3 or ConA. When stimulated by CVB3, the splenocytes from mice of protein group showed significantly stronger proliferative activity than that of PBS group and plasmid group. While when stimulated by ConA, the proliferative activity of protein group and protein/plasmid group were both stronger than that of PBS group, but the difference between the two stronger groups was not significant. 6 The splenocytes from plasmid/protein group showed the strongest specific lymphocytic CTL cytotoxic activity, and in contrast was that of PBS group. But the statistics analysis showed that the difference between them was not significant (P>0.05). 7 Up to the 21th day after CVB3 challenge, the survival rates of PBS group, plasmid group, protein group, plasmid/protein group and protein/plasmid group were 0, 8.33%, 33.33%, 33.33%, 33.33%, respectively. Chi-square test indicated that differences between any two groups were significant. It indicated that the survival states of protein group, plasmid/protein group and protein/plasmid group were better than that of PBS group and plasmid group (P<0.05). 8 After 3LD50 CVB3 challenge, the virus titers of blood in protein group, plasmid/protein group and protein/plasmid group were significantly lower than that of the two control groups (P<0.05).Conclusion: 1 The prokaryotic expressing plasmid pET-his/VP1 was constructed successfully. 2 The protein of CVB3 VP1 is expressed in E.coil. 3 After administration of vaccine to BALB/c mice, the neutralizing antibody increased along with the immune times. 4 Specific lymphocytic proliferative activity and CTL cytotoxic activity in immunized mice of protein/plasmid group were stronger than those in mice immunized with others. 5 Subunit vaccine could provide more effective protections for the immunized mice from death after lethal CVB3 challenge.