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Dmaqp 3基因,构建Dmaqp 3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】Dmaqp 3基因PCR扩增片段大小为879 bp,与森林革蜱aqp 3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∶409600,表明该蛋白具有良好的免疫原性。【结论】本试验克隆了Dmaqp 3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。
【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 Dmaqp 3 gene and construct an amino acid sequence phylogenetic tree of the Dmaqp 3 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 Dmaqp 3 gene was 879 bp,with a similarity of 98.37%to the aqp 3 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 rD
作者
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
WU Jun;HE Wenwen;PU Hao;JIN Min;SHI Wenyu;MA Aijun;LUO Tingxiang;YANG Depeng;Bayinchahan;HU Ercha(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Postdoctoral Research Station of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2023年第11期4589-4599,共11页
China Animal Husbandry & Veterinary Medicine
基金
新疆维吾尔自治区科技厅-国际合作专项(2021E01001)
新疆维吾尔自治区自然科学基金面上项目(2022D01A65)
新疆农业大学研究生科研创新计划项目(XJAUGRI2023035)。
