犬弓首蛔虫C型凝集素4的原核表达及组织定位

doi:10.11843/j.issn.0366-6964.2023.04.028

摘要/Abstract

摘要： 旨在探讨犬弓首蛔虫（Toxocara canis，T.canis） C型凝集素4（C-type lectin 4，C-TL-4）的基因特征，并对其组织分布进行研究。根据Tc-ctl-4的基因组数据（GenBank：AF126830.1），以T.canis为模板对Tc-ctl-4全长基因进行扩增，并进行序列分析和多重序列比对；构建Tc-ctl-4/pCold TF原核表达载体，对重组蛋白进行纯化并制备多克隆抗体；同时采用实时荧光定量PCR （quantitative real-time PCR，qRT-PCR）和间接免疫荧光组化检测Tc-ctl-4基因的转录水平和Tc-CTL-4的组织分布。结果显示，Tc-ctl-4基因的全长序列为842 bp，共编码280个氨基酸，多重序列比对显示，犬弓首蛔虫和猫弓首蛔虫的C型凝集素均含有高度保守的CTLD结构域；SDS-PAGE检测发现重组蛋白Tc-CTL-4的大小约为86 ku，以可溶性形式表达；利用Ni-NTA亲和层析柱纯化蛋白，以500 mmol·L^-1咪唑进行洗脱时可获得高纯度的目的蛋白；将纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体，间接ELISA结果显示，抗体滴度>1：160 000，表明重组蛋白的免疫原性较好；SDS-PAGE结果显示，纯化后的抗体纯度高于95%，Western blot结果显示，抗体能与Tc-CTL-4蛋白特异性结合，表明抗体特异性高。qRT-PCR结果显示，Tc-ctl-4基因在T.canis雌虫子宫和雄虫贮精囊的转录水平最高。间接免疫荧光组化结果显示，Tc-CTL-4主要分布在T.canis生殖组织和体壁，其中雌虫中Tc-CTL-4主要分布在子宫、输卵管和体壁；雄虫中Tc-CTL-4主要分布在输精管和体壁。综上表明，Tc-CTL-4可能在犬弓首蛔虫的繁殖和免疫中发挥了重要作用。

关键词: 犬弓首蛔虫, C型凝集素4, 原核表达, 多克隆抗体, 组织分布

Abstract: The aim of this experiment was to determine the characteristics and tissue distribution of the Ctype lectin 4(Tc-ctl-4)gene of Toxocara canis. The full-length gene of Toxocara canis C-type Lectin 4 (Tc-ctl-4) was amplified as template according to the genomic data of Tc-ctl-4(AF126830.1) and sequence analysis and multiple sequence alignment were performed. The prokaryotic expression vector Tc-ctl-4/pCold TF was constructed, the recombinant protein was purified and polyclonal antibody was prepared. The transcriptional level of Tc-ctl-4 and the tissue distribution of Tc-CTL-4 were detected by quantitative real-time PCR (qRT-PCR) and indirect immunofluorescence histochemistry. The results showed that the full-length sequence of Tc-ctl-4 gene was 842 bp, encoding 280 amino acids.A conserved Ctype lectin like domain (CTLD) was identified in multiple sequence alignments. SDS-PAGE showed that the recombinant protein Tc-CTL-4 was about 86 ku in size and expressed in soluble form. The protein was purified by Ni-NTA affinity chromatography, and high purity protein was obtained by elution with 500 mmol·L^-1 imidazole, which was used to immunize the New Zealand white rabbits to prepare polyclonal antibody. Indirect ELISA showed that the antibody titer was>1:160 000, indicating that the recombinant protein had good immunogenicity and SDS-PAGE showed that the purity of the purified antibody was higher than 95%. Western blot showed that the antibody could specifically bind to Tc-CTL-4, indicating that the antibody was highly specific. The qPCR results showed that the transcription level of Tc-ctl-4 was the highest in the uterus of female worms and the seminal vesicle of male worms. Indirect-fluorescence immunohistochemical assays were carried out to detect the tissue distribution of Tc-CTL-4, which showed predominant distribution of Tc-CTL-4 in the reproductive tissues of adult T. canis, such as in the uterus, oviduct of adult female T. canis and in the vas deferens of adult male T. canis, and body wall of T. canis. It is suggested that Tc-ctl-4 might play an important roles in the reproduction and immunity of T. canis.

Key words: Toxocara canis, C-type lectin 4, prokaryotic expression, polyclonal antibody, tissue expression

中图分类号:

S852.731

王冰楠, 孙雪, 贾红菓, 谭纯, 梅四鹏, 周荣琼. 犬弓首蛔虫C型凝集素4的原核表达及组织定位[J]. 畜牧兽医学报, 2023, 54(4): 1641-1651.

WANG Bingnan, SUN Xue, JIA Hongguo, TAN Chun, MEI Sipeng, ZHOU Rongqiong. Prokaryotic Expression and Tissue Localization of C-type Lectin 4 from Toxocara canis[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1641-1651.

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