边缘革蜱aqp3截短基因的表达、生物信息学分析与多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.11.027

Abstract

Abstract: 【Objective】 This study was aimed to explore and detect the immunogenicity of aquaporin 3 in Dermacentor marginatus (DmAQP3), and provide materials for the subsequent development of anti-tick immunity test.【Method】 PCR technology was used to amplify the Dmaqp3 gene and construct an amino acid sequence phylogenetic tree of the Dmaqp3 gene.The bioinformatics analysis of DmAQP3 protein was carried out.The best region of antigenicity was selected to construct the truncated recombinant plasmid pET-32a-jdDmaqp3, and the recombinant protein DmAQP3 (rDmAQP3) was expressed.The reactogenicity of the recombinant protein was detected by Western blotting.Kunming mice were immunized with the purified recombinant protein rDmAQP3 to prepare polyclonal antibodies, and the titer of polyclonal antibodies was detected by indirect ELISA.【Result】 The PCR amplification fragment size of Dmaqp3 gene was 879 bp, with a similarity of 98.37% to the aqp3 gene of Dermacentor silvarum (XM_049662329.1).The amino acid sequence of the DmAQP3 protein was closest to Dermacentor silvarum (AQP-9 subtype X2) and Dermacentor andersoni (AQP-9 like subtype X2).DmAQP3 was a stable protein with a theoretical isoelectric point of 8.65, it had 6 transmembrane regions and 2 Asn-Pro-Ala (NPA) structures.There were 7 B cell epitope and 18 phosphorylation sites, which was hydrophobic protein.The secondary structure of the protein consisted of alpha helix, beta turn, random coil and extended chain, which accounted for 31.27%, 3.58%, 40.07% and 25.08%, respectively.The tertiary structure prediction showed that the protein was composed of 4 subunits.The truncated recombinant plasmid pET-32a-jdDmaqp3 was successfully constructed and the recombinant protein rDmAQP3 was obtained.Western blotting results showed that the recombinant protein rDmAQP3 reacted with positive serum with a target band of 27 ku in size, indicating that the protein had good reactogenicity.The results of indirect ELISA showed that the titer of the prepared polyclonal antibodies against recombinant protein rDmAQP3 was as high as 1:409 600, indicating that the protein had good immunogenicity.【Conclusion】 In this experiment, Dmaqp3 gene was cloned, the prokaryotic expression vector of DmAQP3 was constructed, and the recombinant protein rDmAQP3 was induced and expressed.The protein had good reactogenicity and immunogenicity, which provided conditions for further study of its biological characteristics and establishment of model animal immune test against ticks.

Key words: Dermacentor marginatus; truncated gene; recombinant protein; Western blotting; polyclonal antibody

CLC Number:

S852.74^＋6

WU Jun, HE Wenwen, PU Hao, JIN Min, SHI Wenyu, MA Aijun, LUO Tingxiang, YANG Depeng, Bayinchahan, HU Ercha. Expression, Bioinformatics Analysis of aqp3 Truncated Gene of Dermacentor marginatus and Preparation of Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4589-4599.

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Expression, Bioinformatics Analysis of aqp3 Truncated Gene of Dermacentor marginatus and Preparation of Polyclonal Antibody

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