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Prior to 1976 only Japanese encephalitis virus (JEV) genotype III could be detected in China. Recently, numerous genotype I JEV strains have been isolated from JE patients, mosquitoes and pigs while genotype III strains remain present. Two kinds of JEV vaccines are currently used in China for the prevention disease: the JE live attenuated vaccine (LAV) SA14-14-2 virus and the inactivated P3 strain (IPV) vaccine. The SA14-14-2 and P3 viral strains were isolated in the year of 1953 and 1949 respectively and both belonged to the JEV genotype III. In order to evaluate the protective efficacy of both vaccines against the JEV genotype I isolates we conducted vaccination-challenge protection assays in mice. These data demonstrated that both LAV (≥ 234 pfu virus) and IPV (1:5 dilution) vaccines effectively conferred protection against all 16 isolates tested following intraperitoneal (i.p.) challenge. However, when vaccinated mice were challenged via intracerebral (i.c.) injection, ≥ 60% LAV vaccinated animals were protected against challenge with most JEV isolates but only ≤ 40% protection was observed following vaccination with IPV. These results indicated that JE vaccines used in China still protected effectively against both JEV genotypes now prevalent in China and that the LAV formulation conferred higher levels of protection compared to the protection conferred by IPV.

A system for the expression of recombinant lumbrokinase (rPI239) was developed in the yeast Pichia pastoris. A total supernatant protein content of 0.174 g/L of high density fermentation broth was obtained. The rPI239 exhibited in vitro fibrinolytic activity. The in vivo activity of rPI239 was measured by prothrombin time, kaolin part thrombin time, thrombin time, and fibrinolytic activity. This work presents the high-density fermentation of rPI239 from P. pastoris and shows that the recombinant protein has similar fibrinolytic activity both in vivo and in vitro.

Nucleocapsid protein plays a critical role in SARS-CoV pathogenesis, and high-level anti-nucleocapsid antibodies are detected in the patients infected by severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Several studies have shown that there exists an interaction between nucleocapsid (N) and membrane (M) protein. In this paper, we investigate whether the expression of membrane protein can affect the immune responses induced by nucleocapsid DNA immunization. Two recombinant plasmids containing M and N coding sequence were constructed. Moreover, in order to get the antigen for ELISA and in vitro stimulation assay, N protein were expressed and purified from E. coli bacteria. Injection of 20mug of the mixture of pVAX1-M and pVAX1-N into the Balb/c mice could elicit the humoral and cellular responses. The ELISA analysis using the N antigen or inactivated SARS-CoV particles as capture antigen showed that co-injection of SARS-M could enhance N-induced antibody production, especially IgG2a subclass. After lymphocytes were stimulated with 10mug/ml purified N antigen, The CD4+ and CD8+ T cells of N and M plus N group were increased compared with those of control groups, and the M protein could augment the activation of lymphocytes induced by N DNA vaccine. Cytokine ELISA analysis revealed that co-injection of M could enhance the levels of IFN-gamma, IL-2 release induced by N antigen. Further experiments in field mouse also support the claim that membrane protein can augment the N-specific immune responses. Virus challenge test was conducted in BSL3 bio safety laboratory with Brandt's vole SARS-CoV model, and the results indicated that co-immunization of M and N antigens could reduce the mortality and pathological changes in lung from the virus infection.

The expression of the hemagglutinins of Avian influenza virus H5 H7and H9 subtypes was studied in this article by Pichia pastoris, one of the eukaryotis expression systems. Three reconstructed expression plasmids and engineering strains, named pPIC9K-H5HA, pPIC9K-H7HA, pPIC9K-H9HA and GS115/pPIC9K-H5HA, GS115/pPIC9K-H7HA, GS115/pPIC9K-H9HA repectively, were obtained. The reconstructed yeast engineering strains were identified by MD and MM plate selecting and PCR. The induced interests proteins were examined by SDS-PAGE and Western-bloting,the results showed that the interest genes were expressed exactly. And this will be helpful in the future study of antigen detection and antibody detection kit, as well in the subunit vaccines developing.