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Aim: To construct and express the gene encoding hIL-6/GM-CSF fusion protein., Methods: The genes encoding the two hIL-6/GM-CSF fusion proteins were constructed in pBV220 expression vector. Fusion proteins were expressed in E.coli BL-21 and purified through Q Sepharose HP ion exchange chromatography and Sephacryl S-200 gel filtration columns. The biologic activities of the fusion proteins were detected by proliferation of hIL-6 dependent cell line B9 and hGM-CSF dependent cell line TF1 with MTT assay., Results: Both fusion proteins were expressed in E.coli BL-21 in the form of inclusion body. The expression levels were more than 25% of the total cell lysates. Both fusion proteins were obtained with high purity which had both hIL-6 and hGM-CSF biologic activities., Conclusion: Two hIL-6/GM-CSF fusion proteins with high purity and bilolgic activities have been acquired successfully.

Objective: To construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation., Methods: The HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10., Results: Both groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response., Conclusions: The adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.

Objective: To observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta)., Methods: Twelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR., Results: Hepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus., Conclusions: The recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.

The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P. ion exchange chromatography after renaturation by dilution. To solve this problem, a novel purification and refolding strategy was adopted. Inclusion bodies was first purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200. Renatured fusion protein was obtained in a purity of more than 95%. It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80%. By the whole procedure, refolding and purification of recombinant protein can be performed within one day. This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E. coli.