猪丁型冠状病毒S1基因的克隆、原核表达及多克隆抗体制备

doi:10.11843/j.issn.0366-6964.2018.06.018

Abstract

Abstract:

In order to obtain polyclonal antibody against porcine deltacoronavirus (PDCoV) spike-1(S1) protein, we cloned the gene (1-1 566 bp), and then expressed the Sa protein (106-1 290 bp) of S1 gene epitope prediction region successfully. The S1 gene epitope prediction region Sa (106-1 290 bp) was cloned into the prokaryotic expression vector pET22b(+) to construct recombinant plasmid pET22b-Sa by using the bioinformatics software to analyze the S1 nucleotide and the amino acid sequence. The recombinant expression vector was transformed into Rosetta (DE3)competent strain, and the recombinant protein was acquired by induction of IPTG. The recombinant protein was purified by ultrafiltration column concentration and immobilized Ni ion affinity chromatography. The activity of the protein Sa obtained was examined by Western blot. New Zealand rabbits were immunized with the Sa protein 4 times to prepare polyclonal antibody. The results showed that the open reading frame of S1 gene of CHN-2015 strain was 1 566 bp (GenBank No.:KY398009), encoding 522 amino acids. It is a non-transmembrane protein with hydrophilicity and 15 potential B cell epitopes Bit;The recombinant protein was expressed in prokaryotic expression. The protein was mainly expressed in soluble form at 37℃ for 5 h at the final concentration of 0.8 mmol·L^-1 IPTG. Immunoblotting results showed that the expressed Sa protein had good immunogenicity in the supernatant and inclusion bodies. The rabbit anti-Sa antibody titer was up to 1:10 240. The results showed that the expressed Sa protein contained antigen epitope recognition region of porcine Deltacoronavirus, and had a good immunogenicity.

Key words: porcine deltacoronavirus, S1 gene, clone, prokaryotic expression, antigenicity, polyclonal antibody

CLC Number:

S852.659.6

LI Cheng, ZHANG Yu-di, HUANG Xiao-bo, LIU Hao-yu, LIU Zhi-peng, ZHAO Yu-jia, CAO San-jie, WEN Xin-tian, WEN Yi-ping, ZHAO Qin, WU Rui. Cloning and Prokaryotic Expression of Porcine Deltacoronavirus S1 Gene and Preparation of Polyclonal Antibody[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1256-1264.

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Cloning and Prokaryotic Expression of Porcine Deltacoronavirus S1 Gene and Preparation of Polyclonal Antibody

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