Establishment Of Model Of Virus - Induced Hepatitis Of Guohoma And Establishment Of Human Enterovirus 71 Recombinant Vaccine

Objective Woodchuck hepatitis virus (WHV) was described initially in1977at the Penrose Zoo in Philadelphia in a colony of woodchucks where high rates of chronic hepatitis and HCC had been observed. Recent studies of the host response of woodchucks to WHV infection and therapy have revealed numerous parallels to the immuno-pathogenesis of HBV infection. Experimental infection of neonatal woodchucks with WHV7P1usually results in a60%-75%frequency of chronic carriers and a25%-40%frequency of naturally recovered infections. Experimental infection of woodchucks with WHV is well-accepted model for the pathogenesis of human HBV infection. Hepatitis B virus infection (HBV) is a major medical problem in China. The lack of a appropriate animal model in China is recognized as an obstacle for research on HBV in China.Method To establish a woodchuck model of hepatitis virus infection, two groups of one-month-old woodchucks were infected with WHV (5×106WID50each animal for the lower dose and2.5×107WID50each animal for the higher dose) via external jugular vein, respectively, and the woodchuck injected with equal volume of saline was used as control. Serum were obtained from woodchucks at different time points (2week,4weeks8week,16week and24week post infection) to detect the WHV DNA by Real-Time PCR, and WHV markers including WHsAg and WHcAb were detected by ELISA. The AST and ALT were monitored by a biochemical analyzer. Liver specimens were sampled by needle biopsy for pathology and immunohistochemistry examination.Result Our results demonstrated that the copies of WHV DNA in serum increased since2weeks and reached a peak at8weeks post infection, consistently, the level of hepatic enzyme were increased accompanied with the appearance of WHsAg and WHcAb in both two groups of woodchucks. The acute hepatitis symptoms, including small hepatocellular necrosis with inflammatory cell infiltration, and scattered hemorrhage were observed in the liver of infected-animal. Then, the copies of WHV DNA in the lower dose group were gradually reduced. At24weeks post infection, the WHV DNA, WHsAg and WHcAb were not detected in the lower dose group and the level of hepatic enzyme reduced and reached the value of uninfected animal, which indicated the acute hepatitis was self-limited. Contrarily, the copies of WHV DNA was maintained at106copies/mL, and the value of WHV DNA copies, WHsAg, WHcAg, AST and ALT were reduced than that of acute hepatitis but obviously higher than that of the uninfected animal, which indicated the acute hepatitis was progressed to chronic hepatitis.Conclusion These results indicated that the woodchucks were successfully infected with WHV. The effect of virus doses on acute hepatitis progressed to chronic hepatitis suggested that the results of WHV infection was not only decided by the ages of woodchucks but also was related to the doses of WHV upon infection. Objective Human Enterovirus71mostly affects children and causes hand, foot and mouth disease with neurological and systemic complications. Due to the lack of specific therapeutic interventions and effective prevention and controlling strategies, there is a serious need for the research and development of preventive vaccines which should be the most efficient approach for preventing and controlling EV71epidemics. The traditional EV71vaccines research was focused on the whole virus vaccines. However, EV71pathogenesis is not yet clear, therefore, the whole virus vaccine may be bring some security issues. Besides, Traditional vaccine antigens have many shortcomings such as adverse reactions, so it is necessary to carry out additional researches about safety, positive protection and easy-producing of the recombinant vaccine candidate. In previous work, our group combined four peptides of virus with no potential neurotoxicity and conflict protection on neonatal mice against EV71infection. In present study, we tested to develop the fermentation and purification procedure of vaccine candidates, which will provide product basis for further pre-clinical study.Method we used the E. coli BL21(DE3) carrying the expression plasmid to express the recombinant protein. At first,The E. coli BL21(DE3) were cultured in shake flasks in LB medium. After induced via IPTG, bacteria were harvested and cracked by ultrasound to identified weather the target protein was soluble. Then we purified inclusion bodies via urea solubilization and gradient renaturation to establish a gradient of fermentation production process in flasks. Then we used10L fermenter contained5L LB medium to ferment E. coli BL21(DE3), After the induction of IPTG, the bacteria were harvested and sonicated to get the inclusion body protein. The inclusion body protein was dissolved in urea and purified through anion exchange column and the gradient renaturation as well as gel filtration purification process steps. Finally the target protein was tested by SDS-PAGE and identified via mass spectrometry.Result Preliminary fermentation experiments showed that the target protein was expressed in the form of insoluble inclusion bodies in Shake flask. After purified and refolded, the protein can be dissolved in PBS. The yield of protein reached20mg/L and the purity of protein was over95%via the detection of SDS-PAGE. Then we established the production process in10L fermenter contained5L LB which included the process of ultrasonication, the purification of inclusion body protein, the solubilization in urea, the purification of anion exchange column, the renaturation gradients and the purification of gel filtration etc., The product was identified to be EV71recombinant protein by mass spectrometry which reached the purity of over95%and the yield of20mg/L.Conclusion The experiment established the preliminary production and purification process of EV71vaccine recombinant protein for neutralization test and laid the foundation for production and purification process according with GMP standard request.