猪腺病毒3型Protease蛋白在大肠杆菌中的表达及其抗体制备

doi:10.16431/j.cnki.1671-7236.2019.06.024

Abstract

Abstract:

This study was aimed to investigate the expression of porcine adenovirus type 3 (PADV3) Protease protein in Escherichia coli (E.coli),and prepare the polyclonal antibody of Protease protein.PADV3 Protease gene was amplified by PCR,the recombinant prokaryotic expression vector pET28a-PADV3-Protease and eukaryotic expression vector pEGFP-PADV3-Protease were constructed and identified by double enzyme digestion and sequencing.The recombinant prokaryotic expression vector pET28a-PADV3-Protease was transformed into E.coli BL21(DE3),the positive recombinant prokaryotic expression strain was induced by IPTG,and identified by SDS-PAGE and Western blotting.The recombinant Protease protein was purified and emulsified with adjuvant to prepare the immunogen which was innoculated into rabbit to prepare a polyclonal antibody against Protease protein.The eukaryotic expression vector pEGFP-PADV3-Protease was transfected into HEK293 cells and the EGFP-protease fusion protein expression stable cell line was screened by G418.The immunological activity and antibody titer of the antibody were detected by immunoperoxidase monolayer staining (IPMA) base on the fusion protein expression stable cell line.The results showed that the length of Protease gene open reading frame (ORF) was 615 bp.Western blotting analysis result showed that Protease protein in the prokaryotic expression system existed as an inclusion body with a molecular weight of 23 ku which was similar to Protease protein expressed in eukaryotic cells.In the fusion protein expression stable cell line,Protease was distributed in both the nucleus and cytoplasm.The Protease specific polyclonal antibody specifically reacted with the EGFP-Protease fusion protein expression stable cell line.The Protease prokaryotic expression strain and the eukaryotic expression cell line were successfully constructed,the PADV3-Protease protein polyclonal antibody was prepared and there was wonderful immunoactivity,providing basic materials for further study of the biological function of Protease protein and the serological diagnostic of PADV3.

Key words: porcine adenovirus type 3(PADV3); Proteasegene; expression; polyclonal antibody

CLC Number:

S852.65⁺9.1

WU Siwen, HU Cong, TANG Qinghai, HAN Taotao, LIU Zhenzhen, XIANG Dongmei, WANG Shushu, LI Yan, YANG Hai, WANG Fangyu. Expression and Antibody Preparation of Porcine Adenovirus Type 3 Protease Protein in Escherichia coli[J]. , 2019, 46(6): 1764-1773.

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Expression and Antibody Preparation of Porcine Adenovirus Type 3 Protease Protein in Escherichia coli

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