红嘴鸥TLR7蛋白多克隆抗体制备及在DF-1细胞中的表达特征分析

doi:10.16431/j.cnki.1671-7236.2023.07.026

Abstract

Abstract: 【Objective】 This study was aimed to prepare the polyclonal antibody of Toll-like receptor 7(TLR7) protein of Larus ridibundus, and analyze the expression characteristics of TLR7 protein in DF-1 cells, so as to provide materials for the in-depth understanding of the function and mechanism of TLR7 protein in Larus ridibundus.【Method】 Primers were designed to amplify the CDS region of TLR7 gene in Larus ridibundus, and its prokaryotic and eukaryotic expression vectors were constructed.The recombinant prokaryotic expression plasmid pET32a-TLR7 was converted to Escherichia coli Transetta (DE3) competent cells for in vitro induction of recombinant protein expression, and the conditions for inducing temperature, inducing time and IPTG concentration were optimally screened.New Zealand White rabbits were choosed to prepare polyclonal antibody, the serum antibody valence was detect by indirect ELISA method, and the antibody specificity was identified by Western blotting.The eukaryotic expression plasmid pcDNA3.1-TLR7 was overexpressed in DF-1 cells, and the expression characteristics of TLR7 protein in Larus ridibundus at different time points after transfection of DF-1 cells were detected by indirect immunofluorescence technology.【Result】 The CDS region of TLR7 gene in Larus ridibundus was 1 182 bp.The double digestion results showed two sets of fragments of different sizes of 5 900 and 1 182 bp (prokaryotic expression vectors), 5 428 and 1 182 bp (eukaryotic expression vector), respectively.Sequencing results showed that the prokaryotic and eukaryotic expression vectors were successfully constructed.When the induction temperature in vitro was 30 ℃ and the final concentration of IPTG was 1.0 mmol/L, a large number of recombinant proteins in the form of inclusion bodies were obtained by culture for 5 h, and the protein size was 63 ku.The antiserum valence of indirect ELISA method was more than 1:256 000.The Western blotting assay results showed that the prepared polyclonal antibody had good specificity.The indirect immunofluorescence results showed that green fluorescence could be observed 2 h after transfection of DF-1 cells, the green fluorescence density gradually increased with time, most of the green fluorescence gathered in and around the nucleus, and a small part was scattered in the cytoplasm.【Conclusion】 The prokaryotic expression of TLR7 protein in Larus ridibundus had good immunogenicity, the prepared polyclonal antibody had high reactivity and specificity, and TLR7 protein was successfully expressed in DF-1 cells.

Key words: Larus ridibundus; TLR7 protein; polyclonal antibody; DF-1 cells; expression

CLC Number:

S852.4⁺3

REN Shengjie, SHEN Hongli, XIANG Xun, DAI Feiyan, ZHU Maoyin, WU Jiali, HU Zhihui, DUAN Gang, CHANG Hua. Polyclonal Antibody Preparation of TLR7 Protein and Its Expression Characteristics Analysis in DF-1 Cells of Larus ridibundus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2865-2875.

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Polyclonal Antibody Preparation of TLR7 Protein and Its Expression Characteristics Analysis in DF-1 Cells of Larus ridibundus

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