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

摘要/Abstract

摘要： 【目的】挖掘和检测边缘革蜱（Dermacentor marginatus）水通道蛋白-3（DmAQP3）的免疫原性，为后续研发免疫抗蜱试验提供材料。【方法】利用PCR技术扩增Dmaqp3基因，构建Dmaqp3基因氨基酸序列系统发育树。对DmAQP3蛋白进行生物信息学分析，截选抗原性最佳区域构建截短重组质粒pET-32a-jdDmaqp3，表达DmAQP3重组蛋白（rDmAQP3），Western blotting检测重组蛋白的反应原性。将纯化的重组蛋白rDmAQP3免疫昆明小鼠，制备多克隆抗体，间接ELISA方法检测多克隆抗体效价。【结果】Dmaqp3基因PCR扩增片段大小为879 bp，与森林革蜱aqp3基因（XM_049662329.1）相似性为98.37%，DmAQP3蛋白氨基酸序列与森林革蜱（AQP-9亚型X2）及安氏革蜱（AQP-9样亚型X2）亲缘关系最近。DmAQP3蛋白理论等电点为8.65，是一种稳定蛋白；具有6个跨膜区及2个天冬酰胺-脯氨酸-丙氨酸（Asn-Pro-Ala，NPA）结构；有7个B细胞抗原表位，18个磷酸化位点，为疏水性蛋白；二级结构由α-螺旋、β-转角、无规则卷曲及延伸链组成，占比分别为31.27%、3.58%、40.07%和25.08%；三级结构预测显示该蛋白由4个亚基组成。成功构建截短重组质粒pET-32a-jdDmaqp3并获得重组蛋白rDmAQP3。Western blotting结果显示，重组蛋白rDmAQP3与阳性血清反应出现大小为27 ku的目的条带，表明该蛋白具有良好的反应原性。间接ELISA结果显示，制备的重组蛋白rDmAQP3多克隆抗体效价高达1：409 600，表明该蛋白具有良好的免疫原性。【结论】本试验克隆了Dmaqp3基因，构建了DmAQP3蛋白原核表达载体，诱导表达出重组蛋白rDmAQP3，该蛋白具有良好的反应原性和免疫原性，为进一步研究其生物学特性及建立模式动物免疫抗蜱试验提供了条件。

关键词: 边缘革蜱; 截短基因; 重组蛋白; Western blotting; 多克隆抗体

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

中图分类号:

S852.74^＋6

伍军, 何文文, 普浩, 金敏, 石文玉, 马爱军, 罗亭祥, 杨德鹏, 巴音查汗, 呼尔查. 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[J]. 中国畜牧兽医, 2023, 50(11): 4589-4599.

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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