Abstract: 【Objective】 The purpose of this study was to construct a recombinant Adenovirus with VP4 gene of Porcine rotavirus(PoRV) epidemic strain PoRV G9P[23] in China, and lay a preliminary foundation for the development of candidate engineering vaccine of PoRV.【Method】 PoRV VP4 gene was synthesized by referring to the VP4 gene sequence of PoRV G9P[23] strain in GenBank (accession No.:MH898990.1). The obtained target gene was recombined with Adenovirus shuttle vector pAdTrack-CMV and transferred into Escherichia coli Top10 competent cells to construct Adenovirus shuttle vector pAdTrack-CMV-VP4. The recombinant plasmid pAd-VP4 was obtained by homologous recombination with BJ5183 competent cells containing adenovirus backbone pAdEasy-1 after linearized by PmeⅠ endonuclease. The recombinant plasmid was digested by PacⅠ enzyme and transformed into Escherichia coli DH5α competent cells. The recombinant plasmid was transfected into HEK293A cells to obtain recombinant Adenovirus rAd-VP4. The recombinant Adenovirus was expanded and the half tissue culture infective dose (TCID₅₀) of recombinant Adenovirus was determined.RT-PCR was used to detect its expression in vitro, and its reactivity was detected by Western blotting. The prepared recombinant Adenovirus was inoculated mice intraperitoneally with different virus titers and different times of immunization. The serum was collected to determine IgG antibody levels by ELISA kit.【Result】 A recombinant Adenovirus rAd-VP4 with a size of 2 343 bp was amplified by RT-PCR, and the sequencing results were correct, indicating that the recombinant Adenovirus rAd-VP4 was successfully constructed. The viral titer of rAd-VP4 was 10^6.5 TCID₅₀. The recombinant Adenovirus rAd-VP4 was correctly expressed at the protein level and the molecular mass of the protein was about 87 ku. The results of antibody detection showed that the serum IgG antibody level of mice immunized with 10^6.5 TCID₅₀ rAd-VP4 was significantly higher than that of 10^5.3 TCID₅₀ TGE-PED-PRV triple live vaccine on the 35th and 42nd day(P<0.05). The antibody level of 10^6.5 TCID₅₀ rAd-VP4 on the 35th and 42nd day after immunization was significantly higher than that of 10^5.5 and 10^4.5 TCID₅₀ rAd-VP4 (P<0.05). The antibody level of 10^5.5 TCID₅₀ rAd-VP4 on the 28th day after immunization was significantly higher than that of 10^6.5 and 10^4.5 TCID₅₀ rAd-VP4 (P<0.05). There was no significant difference in IgG antibody produced by 10^6.5 TCID₅₀ rAd-VP4 immunizing 2 and 3 times at different immunization times (P>0.05).【Conclusion】 In this study, the recombinant Adenovirus rAd-VP4 was successfully constructed, and it's virus titer was 10^6.5 TCID₅₀. 10^6.5 and 10^5.5 TCID₅₀ rAd-VP4 produced higher IgG antibody levels on the 42th and 28th day, respectively. There was no significant difference in IgG antibody level between the second immunization and the third immunization. The results could provide reference for the development of PoRV recombinant Adenovirus vaccine candidate.

Key words: Porcine rotavirus(PoRV); recombinant Adenovirus vector; IgG antibody level