High Expression and Immunogenicity Evaluation of Porcine parvovirus VP2 Gene in Silkworm (Bombyx mori)
ZHAN Peng-Fei1, DOU Jin-Ping2, YI Yong-Zhu3, LIU Xing-Jian2, LI Yi-Nyu2, FENG Shi-Min1, ZHANG Zhi-Fang2, ZHANG Jin-Wei1
1 Huzhou Agricultural Science and Technology Development Center, Huzhou 313000, China; 2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3 School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China
Abstract：： Porcine parvovirus (PPV) can cause porcine (Sus scrofa domesticus) reproductive disorder. Vaccination is an effective method to prevent pigs from PPV disease and improve the immunity and reproductive rate of sows. In order to efficiently express PPV structural protein VP2 gene and prepare PPV genetic engineering subunit vaccine, the PPV-VP2 gene was optimized and synthesized according to silkworm (Bombyx mori) codon frequency in this study. The VP2 gene was cloned to transfer vector and recombined into the parental virus BmBacmid with orf1629 gene deficient by co-transfection. The recombinant baculovirus rBmNPV (PPV-VP2) was obtained and used to infect silkworm. The expressed antigen was used to prepare subunit vaccine, and guinea pigs (Cavia porcellus) were used for vaccine efficacy evaluation. The expressed recombinant protein was confirmed by Western blot and mass spectrometry. The observation of electron microscope indicated that the virus like particles (VLP) could be self-assembled into a similar size and morphology to PPV. The results of hemagglutination assay (HA) indicated that expressed antigen titer per gram of silkworm pupae was equivalent to more than 200 doses of commercial vaccine. Guinea pigs were immunized with 3 different doses of PPV-VP2 antigen contents, all inoculated animals had a seroconversion, and the antibody could neutralize PPV in vitro. If a guinea pig was immunized with 5 mg of silkworm pupae (parallel to 200 doses of antigen in commercial vaccine), the hemagglutination inhibition (HI) and neutralizing
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