1株猪腺病毒3型的分离培养及其fiber蛋白多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.02.024

Abstract

Abstract: The purpose of this study was to isolate and culture the pathogen of a clinical diarrheal disease and to explore the biological characteristics of the main antigen proteins encoded by the pathogen.The nucleic acid of Porcine adenovirus type 3 (PADV3) was detected by nested PCR and the virus was isolated and cultured,and was identified by immunoperoxidase monolayer staining (IPMA).The ORF region of fiber gene was amplified by PCR and cloned into pET-28a(+) vector and transformed into E.coli BL21 (DE3) competent cells.Recombinant fiber protein (recfiber) was induced by IPTG and was identified by SDS-PAGE and Western blotting.The purified recombinant protein was immunized to rabbits to prepare polyclonal antibody and determine its immune activity.The results showed that the pathogen was positive for PADV3 nucleic acid,and infected ST cells produced typical "grapevine" cytopathic effect (CPE).The nucleic acid of PADV3 was stable in the passages from P5 to P11,and the P5 of the isolated strain was serologically positive for PADV3.The open reading frame (ORF) of fiber gene was 1 296 bp (GenBank accession number:MT774498),which had the highest nucleotide similarity (86.1%) with PADV3 strain and less than 40% similarity with other strains.The molecular weight of the recombinant fiber protein expressed in prokaryotic cells was about 45.2 ku.The polyclonal antibody of fiber protein showed specific nuclear staining with PADV3 infected cells.A PADV3 strain named PADV3-HY1812 was successfully isolated in this experiment and the polyclonal antibody against fiber protein had good immune activity.

Key words: Porcine adenovirus type 3; isolation and culture; fiber gene; prokaryotic expression; antibody

CLC Number:

S852.65

CHEN Guoliang, TANG Qinghai, YANG Hai, YANG Can, WANG Fangyu. Isolation and Culture of a Porcine Adenovirus Type 3 Strain and Preparation of Polyclonal Antibody Against Its fiber Protein[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 619-630.

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Isolation and Culture of a Porcine Adenovirus Type 3 Strain and Preparation of Polyclonal Antibody Against Its fiber Protein

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