非洲猪瘟病毒RNA聚合酶亚基D205R基因生物信息学分析及多克隆抗体制备

doi:10.11843/j.issn.0366-6964.2023.05.025

Abstract

Abstract: African swine fever virus (ASFV) pD205R protein is involved in virus gene transcription, which belongs to ASFV RNA polymerase, similar with RPB5 of eukaryotic RNA polymerase II. In this study, in order to further understand the structure, function of pD205R and interaction mechanism between virus and host, the sequence of D205R gene from different ASFV strains deposed in GenBank were analyzed by using DNAStar and Mega7.0 software. Physicochemical Properties and Structure of Protein were predicted by using ExPASy, GOR4 and AlphaFold software, and its prokaryotic expression and preparation of anti-pD205R protein polyclonal antibody were performed. Furthermore, the D205R gene was amplified and cloned into pET-28a vector, named as pET-28a-D205R. The results showed that the D205R gene was highly conserved among different genotypes of ASFV isolates. The physicochemical properties analysis showed that pD205R protein was hydrophilic with poor stability, its molecular formula was predicted as C₁₀₈₈H₁₇₁₂N₂₇₈O₂₉₅S₈. The secondary structure and tertiary structure indicated that α helix was the main structural form, proportion for 35.12%. Furthermore, the pET-28a-D205R recombinant plasmids were constructed successfully using ASFV CN/SC/2019 strain genome as template and transformed into E. coli Rosetta (DE3) competent cells, the recombinant proteins were successfully induced and expressed by IPTG, with the size of 23.3 ku. Then the obtained protein was purified by nickel column affinity chromatography, which could react specifically with ASFV positive serum by Western blot identification. The titer of polyclonal antibody prepared by immunizing New Zealand white rabbits could reach to 1:51 200, and could specially recognize the native pD205R protein expressed in ASFV-infected porcine alveolar macrophages (PAMs) by Western blot and IFA detection. It laid a foundation for further study the function of D205R gene and its role in host-pathogen interaction.

Key words: African swine fever virus, pD205R protein, prokaryotic expression, polyclonal antibodies

CLC Number:

S852.659.1

WANG Guochao, ZHAO Yaru, ZHANG Zhonghui, ZHANG Yulong, BAI Ge, GENG Shuxian, FAN Jie, YANG Jifei, GUAN Guiquan, YIN Hong, LUO Jianxun, NIU Qingli. Bioinformatics Analysis of RNA Polymerase Subunit D205R Gene of African Swine Fever Virus and Polyclonal Antibody Preparation[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2042-2049.

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Bioinformatics Analysis of RNA Polymerase Subunit D205R Gene of African Swine Fever Virus and Polyclonal Antibody Preparation

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