瑶山亚种树鼩ISG15蛋白表达及其多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2022.01.030

Abstract

Abstract: [Objective] The interferon stimulating gene 15 (ISG15) gene of Tupaia belangeri yaoshanensis (T. b. yaoshanensis) was cloned, the ISG15 protein was highly expressed and purify in Escherichia coli, then its polyclonal antibody was prepared, which laid the foundation for its biological application and the establishment of detection methods. [Method] Total RNA was extracted from the peripheral lymphocytes of T. b. yaoshanensis, and ISG15 gene was amplified by RT-PCR. ISG15 gene was subcloned into the eukaryotic expression vector to construct the recombinant eukaryotic expression plasmid pcDNA3.1-ISG15, and transiently transfected into renal cells of Cricetidae baby hamster syrian kidney (BHK-21) cells. At the same time, it was subcloned into the prokaryotic expression vector pET-28a(+) and the recombinant expression plasmid pET-28a-ISG15 was constructed. The pET-28a-ISG15 was transformed into E. coli BL21(DE3), and the ISG15 protein of T. b. yaoshanensis was induced by IPTG. The recombinant protein was purified by nickel ion affinity chromatography and immunized with mice to obtain mouse anti-ISG15 polyclonal antibody, and its reactivity was detected by Western blotting and IFA. [Result] ISG15 gene of T. b. yaoshanensis was successfully cloned, and its eukaryotic and prokaryotic expression vectors were constructed. The eukaryotic expression vector could be highly expressed in BHK-21 cells. The prokaryotic expression vector was induced with 0.5 mmol/L IPTG at 30 ℃ for 6 h to obtain the recombinant protein with molecular weight at approximately 22 ku. The protein was expressed in the form of inclusion body and purified by nickel ion affinity chromatography. The mice were immunized with the emulsified recombinant protein to prepare the polyclonal antibody against ISG15 of T. b. yaoshanensis. Western blotting showed that the mouse-anti-T. b. yaoshanensis ISG15 polyclonal antibody could still bind to 0.01 μg of ISG15 recombinant protein at the dilution of 1:8 000. IFA tests showed that the antibody could react with the proteins overexpressed by eukaryotic cells, with good reactivity. [Conclusion] The constructed prokaryotic expression vector highly expressed ISG15 protein of T. b. yaoshanensis in E. coli, and the recombinant protein had good immunogenicity after purification and renaturation. The obtained polyclonal antibody laid a good foundation for further study on the antiviral infection immunity of T. belangeri.

Key words: Tupaia belangeri yaoshanensis (T. b. yaoshanensis); ISG15; eukaryotic expression; prokaryotic expression; polyclonal antibody

CLC Number:

R392.11

CAO Yingying, LI Huijun, LI Baoying, LIANG Liang, LENG Jing, TANG Haibo. Expression of ISG15 Protein of Tupaia belangeri yaoshanensis and Preparation of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 273-282.

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Expression of ISG15 Protein of Tupaia belangeri yaoshanensis and Preparation of Its Polyclonal Antibody

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