摘要
蜱类水通道蛋白(AQP)是负责胞内外水分子运输的蛋白质,属于内在蛋白超家族成员。该蛋白是一种潜在的抗蜱疫苗候选抗原。为探究边缘革蜱(Dm)AQP(Dm AQP)的生物学特性及其是否具有抗原性,本研究利用PCR扩增Dm两条不同的Dmaqp基因,分别得到582 bp的Dmaqp1基因和1094 bp的Dmaqp2基因,并将其翻译成相应的蛋白,经各种生物信息学软件分析后显示,Dm AQP1和Dm AQP2蛋白理论等电点分别为4.76和8.65,Dm AQP1有4个跨膜区及1个天冬酰胺-脯氨酸-丙氨酸(NPA)结构,Dm AQP2有4个跨膜区以及2个NPA结构;Dm AQP1和Dm AQP2蛋白分别有5个和9个B细胞抗原表位,11个和25个磷酸化位点,两种蛋白均为疏水性蛋白;Dm AQP1和Dm AQP2蛋白二级结构均主要由α-螺旋以及无规则卷曲组成,分别占34.02%、39.69%和32.93%、39.52%。基于Dmaqp1和Dmaqp2基因,从其编码区选择抗原决定簇密集且跨膜区域少的片段进行截短基因的PCR扩增,构建原核表达质粒p ET-32a-jd Dmaqp1和p ET-28a-jd Dmaqp2并转化至大肠杆菌BL21中,重组菌经IPTG诱导获得大小约为28 ku和14 ku的重组蛋白rjd Dm AQP1和rjd Dm AQP2;将纯化的重组蛋白免疫小鼠,制备小鼠多克隆抗体,western blot鉴定结果显示,两种蛋白分别出现了28 ku和14 ku的特异性条带,表明rjd Dm AQP1和rjd Dm AQP2均具有较好的反应原性。本研究首次经原核系统表达了Dm AQP,为进一步研究其生物学特性以及开展后续模式动物免疫抗蜱实验奠定基础。
Tick aquaporins which belong to the inner protein superfamily are responsible for the transport of intracellular and extracellular water molecules.This protein is a potential candidate antigen for anti-tick vaccine.In order to explore the biological characteristics and antigenicity of the aquaporin( Dm AQP) of Dermacentor marginatus(Dm),two different Dmaqp genes of D.marginatus were amplified by PCR.A 582bp Dmaqp1 sequence and a 1094bp Dmaqp2 sequence were obtained and translated into the corresponding proteins.Bioinformatic analysis showed that theoretical isoelectric points of Dm AQP1 and Dm AQP2 were 4.76 and 8.65,respectively.Dm AQP1 has four transmembrane domains and one NPA structures,Dm AQP2 has four transmembrane domains and two NPA structures.Dm AQP1 and Dm AQP2 proteins have 5 and 9 B cell epitopes,11 and 25phosphorylation sites,respectively.Both proteins are hydrophobic proteins.The secondary structures of Dm AQP1 and Dm AQP2proteins are mainly composed of α-helixes and random coils,accounting for 34.02% and 39.69% for Dm AQP1,and 32.93% and39.52% for Dm AQP2.The fragments with continuous B cell epitopes and few transmembrane regions were selected from the CDs for prokaryotic expression.The expression plasmids p ET-32a-jd Dmaqp1 and p ET-28a-jd Dmaqp2 were constructed and transferred into E.coli BL21.Recombinant proteins were obtained from p ET-32a-jd Dmaqp1/BL21 and p ET-28a-jd Dmaqp2/BL21 with sizes of about 28ku and 14ku were induced by IPTG.The purified recombinant fusion protein was used to immunize mice to prepare mouse polyclonal antibody.The results of western blot showed that the two proteins had specific bands,indicating that both rjd Dmaqp1and rjd Dmaqp2 had antigenicity.This study was the first exogenously expressed D.marginatus aquaporin,which would lay the foundation for further study of its biological characteristics and follow-up expriments on model animal immunity against ticks.
作者
伍军
何文文
刘燕
王水怡
普浩
金敏
温丽翠
巴音查汗
呼尔查
WU Jun;HE Wen-wen;LIU Yan;WANG Shui-yi;PU Hao;JIN Min;WEN Li-cui;BAYIN Cha-han;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
《中国预防兽医学报》
CAS
CSCD
北大核心
2023年第7期755-760,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
上海合作组织科技合作伙伴计划(2021E01001)
新疆维吾尔自治区自然科学基金面上项目(2022D01A65)。
作者简介
伍军(1997-),男,安徽合肥人,硕士研究生,主要从事蜱虫生活史及抗蜱疫苗的研究;通信作者:巴音查汗,E-mail:2514062881@qq.com;通信作者:呼尔查,E-mail:121859196@qq.com。
