China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (8): 3336-3344.doi: 10.16431/j.cnki.1671-7236.2023.08.031

• Preventive Veterinary Medicine • Previous Articles    

Construction and Identification of a Recombinant Adenovirus Expressing Rabbit Hemorrhagic Disease Virus Type 2 (RHDV2) VP60 Protein Leaded by Kozak Sequence

XIAO Lu1, YU Jifeng1, LUO Yan2, XIE Jing1, YE Yonggang1, WU Xuejing1, MAO Congjian1, LI Xingyu1, CAO Ye1, LIN Yi1, PAN Meng1, YE Jianqiang1, WEI Yong1, KANG Runmin1   

  1. 1. Sichuan Provincial Key Laboratory of Animal Breeding and Genetics, Sichuan Animal Science Academy, Chengdu 610066, China;
    2. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China
  • Received:2023-03-02 Published:2023-07-27

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Abstract: 【Objective】 The purpose of this study was to construct a recombinant Adenovirus expressing Rabbit gemorrhagic disease virus type 2(RHDV2) VP60 protein leaded by Kozak sequence,based on a replication-defective Human adenovirus type 5(Ad5) and provide a basis for the research of RHDV2 new vaccine.【Method】 The VP60 gene with Kozak sequence were modified,synthesized and cloned to recombinant Adenovirus shuttle plasmid,and transferred into Escherichia coli TOP10 competent cells to construct Adenovirus shuttle vector,and the recombinants were analyzed and identified by restriction enzyme and sequencing.The recombinant Adenovirus Ad5-RHDV2-VP60 was packaged by the site-directed homologous recombination of shuttle plasmid and Adenovirus backbone plasmid,based on HEK293 cells using the Ad Max system.Then,the expression was verified by infecting HEK293 cells,and the recombinant Adenovirus was identified by RT-PCR,Western blotting and indirect immunofluorescence assay (IFA),and the titer of the recombinant Adenovirus was determined by Reed-Muench method.【Result】 Shuttle plasmid digestion result showed two bands with sizes of 3 895 and 1 752 bp,and the sequencing alignment results showed that it shared more than 99% similarity to the corresponding sequences published on GenBank,and the result showed that the shuttle plasmid was successfully constructed.The result of RT-PCR showed the 515 bp specific band,and the Western blotting result showed that the recombinant Adenovirus expressed a 60 ku VP60 protein.IFA results showed that HEK293 cells infected with the recombinant Adenovirus produced green fluorescence,and the VP60 specific protein could express in the HEK293 cells.The results of virus titer determination showed that the TCID50of the primary recombinant Adenovirus was 105.5/0.1 mL.【Conclusion】 This study constructed a recombinant Adenovirus containing RHDV2 VP60 gene,which could successfully express RHDV2 VP60 with good antigenicity,and this study also provided a virus model for further research and development of RHDV2 vaccine.

Key words: rabbit hemorrhagic disease; Rabbit hemorrhagic disease virus type 2(RHDV2); recombinant Adenovirus; HEK293 cells; Kozak sequence

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