To investigate the construction and expression of rBCG combining human granulocytemacrophage colony stimulating factor(GM-CSF) and EB virus (EBV) encoding immediate early gene(BZLF1).Then the antitumor activity and immunological mechanisms of rBCG including fusion genes GM-CSF and BZLF1 were researched. Methods The purified GM-CSF and BZLF1 were amplified by PCR and inserted into plasmid pMV261, then transformed them intoEscherichia coli DH5α(E.coli . DH5α). In LB culture medium containing kanamycin, the positive colony was selected, the correct sequence of the GM- CSF and BZLF1 were connected by the polypeptide (Gly4Ser)3 series with the splicing overlap extension technology. The fusion gene GCBF was constructed and cloned into pMV261, transformed competent bacteria and selected on LB culture medium flat containing kanamycin. Plasmid pMVGCBF extracted from positive clones were transformed into competent BCG. Western- blot was employed for determination of expression of GCBF. C57BL/6 mice were used to build animal models of EB virus-positive tumors. Antitumor activity was analyzed by the formation time of tumors, survival time and tumor weight. The specific antibodies of mice stimulated with rBCG was detected by ELISA, lactate dehydrogenase assay was used to detect the mouse cellular immunity, HE staining of tumor tissue to assay lymphocyte infiltration in tumor of mice,so the rBCG immune effect was researched. At last,we used statistical methods to analized the immunization of rBCG.Results The RT-PCR product sizes of objective gene GM-CSF and BZLF1 were 461bp and 788bp, consistented with expected values. The recombinant plasmid was confirmed by restriction double enzymes，amplification and sequencing ,then the fusion gene (1209bp ) was correctly inserted into the vector.pMVGCBF were correctly transformed into competent BCG.The expression of fusion protein GCBF was detected in rBCG.Tumor formation time was delayed in mice immunized by rBCG.Tumor growth was slow and survived time of mice was prolonged.The mice immunized by rBCG could produce specific IgG antibodies of GM-CSF and BZLF1.At the same time,specific CTL activity was detected in mice and tumor tissue infiltration of lymphocytes was found by microscopy. Conclusion rBCG encoding GCBF fusion gene was successfully constructed to have provided basis for further study of the function of rBCG.The mice immunized by rBCG could produce humoral and cellular immune responses.The mice produced a strong antitumor effect. The EB virus-positive tumors was significantly inhibited in mice.
Yepin Yu has completed his Bachelor degree from Xiamen University and now doctorates in Marine Biology in South China Sea Institute of Oceanology, Chinese Academy of Sciences. He is a Ph D. student of Prof. Qin, the finder of Singapore Grouper Iridovirus (SGIV). Yepin’s academic interests include viral immune evasion and virus-host reation. He has published 3 papers in reputed journalspi
It has been demonstrated that tumour necrosis factor receptor (TNFR) homologues encoded by viruses are usually involved in virus immune evasion by regulating the host immune response or mediating apoptotic cell death. Here, a novel TNFR-like protein encoded by Singapore grouper iridovirus (SGIV VP51) was cloned and characterized. Amino acid analysis showed that VP51 contained three cysteine-rich domains (CRDs) and a transmembrane domain at its C terminus. The expression of VP51 in vitro enhanced cell proliferation, and affected cell cycle progression via altering the G1/S transition. Furthermore, VP51 overexpression improved cell viability during SGIV infection via inhibiting virus-induced apoptosis, evidenced by the reduction of apoptotic bodies and the decrease of caspase-3 activation. In addition, overexpression of VP51 increased viral titre and the expression of viral structural protein gene MCP and cell proliferation promoting gene ICP-18. In contrast, the expression of the viral apoptosis inducing gene, LITAF, was significantly decreased. Although all three CRDs were essential for the action of VP51, CRD2 and CRD3 exerted more crucial roles on virus-induced apoptosis, viral gene transcription and virus production, while CRD1 was more crucial for cell proliferation. Together, SGIV TNFR-like products not only affected cell cycle progression and enhanced cell growth by increasing the expression of the virus encoded cell proliferation gene, but also inhibited virus-induced apoptotic cell death by decreasing the expression of the viral apoptosis inducing gene. Our results provided new insights into understanding the underlying mechanism by which iridovirus regulated the apoptotic pathway to complete its life cycle.