犬弓首蛔虫TcCPG1的表达及其与几丁质的结合特性  

Expression and Chitin-Binding Feature of TcCPG1 of Toxocara canis

作　　者：陈绍基 李芳[1] 尹莎莎 江艾耘 周荣琼[1] CHEN Shaoji;LI Fang;YIN Shasha;JIANG Aiyun;ZHOU Rongqiong(College of Animal Science,Southwest University,Rongchang 402460,China)

机构地区：[1]西南大学动物科学学院,荣昌402460

出　　处：《畜牧兽医学报》2020年第2期356-364,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31672541,31172313)。

摘　　要：为探讨犬弓首蛔虫(Toxocara canis,T.canis)软骨素蛋白多糖TcCPG1的表达特征及其与几丁质的结合方式,为犬弓首蛔虫软骨素蛋白多糖的功能研究提供依据,笔者克隆T.canis软骨素蛋白多糖1基因(Tc-cpg-1)并原核表达重组蛋白TcCPG1,采用镍柱亲和层析技术对重组蛋白进行纯化,并与几丁质进行结合试验;采用实时荧光定量PCR (quantitative real-time polymerase chain reaction, qRT-PCR)进行Tc-cpg-1基因的转录水平的性别和组织差异表达分析。结果显示:Tc-cpg-1基因编码区(coding sequence, CDS)的长度为1 251 bp,共编码417个氨基酸;SDS-PAGE电泳结果显示重组蛋白TcCPG1以包涵体形式存在,相对分子质量大小约为70 ku;对表达条件进行优化后,以0.8 mmol·L-1 IPTG于16℃诱导14 h时可获得大量目的蛋白;利用镍柱亲和层析纯化可以获得具有较高纯度的目的蛋白,与几丁质进行结合试验,验证了重组蛋白TcCPG1能够与几丁质结合;通过qRT-PCR对Tc-cpg-1基因转录水平的组织差异性进行分析,发现Tc-cpg-1在雌虫中高量表达,尤其在雌虫的生殖组织中表达量最高,表明Tc-cpg-1可能参与了T.canis雌虫的生殖过程,推测TcCPG1可能通过与卵壳中几丁质的相互作用从而影响T.canis的胚胎和生殖发育过程。To explore the expression and chitin-binding manner of chondroitin proteoglycans 1 of Toxocara canis(TcCPG1) and to provide a basis for the functional study of this proteins in T. canis. In the present work, the chondroitin proteoglycans 1 gene(Tc-cpg-1)of T. canis was molecularly cloned and expressed in a prokaryotic expression system for recombinant protein TcCPG1, which was then purified using Ni-NTA affinity chromatography and tested in a chitin-binding assay. Quantitative real-time polymerase chain reaction(qRT-PCR) was employed to determine the transcriptional differences of Tc-cpg-1 between sexes and among tissues of T. canis. The results showed that the coding sequence of Tc-cpg-1 was 1 251 bp in length, encoding 417 amino acids. SDS-PAGE analysis showed that the recombinant protein TcCPG1 was expressed in inclusion bodies, and the molecular weight was estimated at ~70 ku. Optimized conditions(induction by 0.8 mmol·L-1 IPTG for 14 h and incubation at 16 ℃) facilitated an efficient production of the recombinant protein TcCPG1. Following Ni-NTA affinity purification, the recombinant protein bound chitin, suggesting a chitin-binding activity of TcCPG1. In addition, differential analysis showed a high transcriptional level of Tc-cpg-1 in female adult worm, particularly in the reproductive tissues, indicating potential roles of Tc-cpg-1 in reproductive process(embryonic and reproductive development) of female worms, which might be achieved by interacting with the chitin in egg shell.

关 键 词：犬弓首蛔虫 CPG1 原核表达 几丁质结合 

分 类 号：S852.731[农业科学—基础兽医学]