高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2023.05.032

Abstract

Abstract: 【Objective】 The purpose of this study was to express the Hexon protein of Fowl adenovirus serotype 4 (FAdV-4) by prokaryotes expressed system and prepare its polyclonal antibody,so as to provide materials for the study of FAdV-4 Hexon function.【Method】 The method accurate synthesis of long DNA sequences based on PCR (PAS) was used to design the full-length spline primer,and protective bases were designed on both ends of the primer to synthesize FAdV-4 Hexon gene.It was connected to the pCzn1-Hexon expression vector by homologous recombination technology,and the pCzn1-Hexon recombinant plasmid was obtained for prokaryotic expression.The purified recombinant protein was used to immunize New Zealand White rabbits to prepare rabbit polyclonal antibody against Hexon protein.The titer of polyclonal antibody was determined by indirect ELISA,and the specificity of polyclonal antibody was identified by Western blotting and indirect immunofluorescence assay (IFA).【Result】 The recombinant prokaryotic expression plasmid pCzn1-Hexon was confirmed to be constructed correctly by double restriction enzyme digestion and sequencing.The obtained recombinant protein was expressed in the form of inclusion body,and its molecular weight was about 41 ku.After immunizing New Zealand White rabbits with purified recombinant protein,the antibody titer was up to 1∶512 000 by indirect ELISA.Using the prepared polyclonal antibody as the first antibody,the expression of Hexon protein in chicken LMH cells was detected by Western blotting and IFA,indicating that the prepared polyclonal antibody could react specifically with Hexon protein.【Conclusion】 The recombinant Hexon protein had been highly expressed in Escherichia coli,and the prepared and purified anti-Hexon polyclonal antibody showed high reactivity and specificity,which laid a foundation for in-depth analysis of the biological function of Hexon protein and its role in FAdV-4 infection and pathogenesis.

Key words: Fowl adenovirus serotype 4; Hexon protein; prokaryothic expression; polyclonal antibody

CLC Number:

S852.659.1

WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.

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Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies

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