猪NLRP6蛋白的原核表达、纯化及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2019.09.026

Abstract

Abstract: The aim of the experiment was to express and purify porcine NLRP6 protein and prepare mouse anti-NLRP6 polyclonal antibody.Specific primers were designed according to the nucleotide sequence of porcine NLRP6 (GenBank accession No.:XM_003124236.4).NLRP6 gene fragment was amplified from pMD-19T-NLRP6 recombinant vector by PCR method,and the amplified product was connected with prokaryotic expression vector pET-32a(+) to obtain recombinant plasmid pET-32a-NLRP6.After identification,PCR and sequencing analysis,it was transformed into competent cells of Escherichia coli Rosetta(DE3),and expressed by IPTG and identified by Western blotting.The recombinant fusion protein was purified by denaturation,nickel affinity purification and renaturation.The recombinant fusion protein was immunized to BALB/c mice to prepare polyclonal antibodies.The results showed that a 576 bp NLPR gene was successfully cloned,and the recombinant plasmid pET-32a-NLRP6 was constructed correctly.The recombinant fusion protein of NLRP6 with a size of about 34 ku was obtained by IPTG induction.Western blotting analysis showed that the recombinant protein was positive for mouse anti-6×His monoclonal antibody.The solubility analysis showed that the recombinant fusion protein of NLRP6 existed in the form of inclusion bodies,accounting for about 95%.The purified NLRP6 recombinant fusion protein was used to immunize BALB/c mice to obtain polyclonal antibodies.Western blotting analysis showed specific reactions.The immunogenic NLRP6 protein and its mouse-derived polyclonal antibodies were successfully prepared in this experiment,which provided basic materials for the study of biological functions of NLRP6 protein and pathogenesis of related diseases.

Key words: porcine NLRP6 protein; cloning; prokaryotic expression; polyclonal antibody

CLC Number:

S828

YANG Wenjing, LI Yupeng, LIANG Dongmei, WANG Qian, YANG Sheng, QIAO Jiayun, YAO Shengning, LI Haihua. Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Porcine NLRP6 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2707-2714.

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Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Porcine NLRP6 Protein

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