广州管圆线虫半胱氨酸蛋白酶AcCBL1和AcCBL2基因的克隆、表达及鉴定

中国寄生虫学与寄生虫病杂志 ›› 2018, Vol. 36 ›› Issue (5): 469-473.

广州管圆线虫半胱氨酸蛋白酶AcCBL1和AcCBL2基因的克隆、表达及鉴定

白慧芳¹, 柯琪文², 陈韵秋¹, 刘芷晴², 张玲敏¹, 吴春云¹, 袁桂秀¹, 詹希美³, 程梅^1,^*()

1 暨南大学基础医学院病原生物学教研室,广州510632
2 暨南大学基础医学院临床医学系,广州510632
3 中山大学中山医学院寄生虫学教研室,广州510080

收稿日期:2018-05-16 出版日期:2018-10-30 发布日期:2018-11-13
通讯作者: 程梅
基金资助:
国家自然科学基金（No. 81401680）;广东省医学科学技术研究基金（No. A2015334）;中央高校基本科研业务费专项资金（No. 21613306）

Cloning, expression and identification of cysteine protease genes AcCBL1 and AcCBL2 of Angiostrongylus cantonensis

Hui-fang BAI¹, Qi-wen KE², Yun-qiu CHEN¹, Zhi-qing LIU², Ling-min ZHANG¹, Chun-yun WU¹, Gui-xiu YUAN¹, Xi-mei ZHAN³, Mei CHENG^1,^*()

1 Department of Pathogen Biology, Medical College, Jinan University, Guangzhou 510632, China
2 Department of Clinical Medicine, Medical College, Jinan University, Guangzhou 510632, China
3 Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China

Received:2018-05-16 Online:2018-10-30 Published:2018-11-13
Contact: Mei CHENG
Supported by:
Supported by the National Natural Science Foundation of China （No. 81401680）, Medical Scientific Research Foundation of Guangdong Province of China （No. A2015334）, and the Fundamental Research Funds for the Central Universities（No. 21613306）

摘要/Abstract

摘要：

目的克隆、表达广州管圆线虫半胱氨酸蛋白酶1（AcCBL1）和AcCBL2基因,分析重组半胱氨酸蛋白酶rAcCBL1和rAcCBL2的免疫反应性。方法基于广州管圆线虫Ⅳ期幼虫的cDNA文库筛选获得AcCBL1和AcCBL2基因,构建pET-28a-AcCBL1和pET-28a-AcCBL2重组质粒,转入大肠埃希菌BL21（DE3）中,经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达、镍柱纯化后,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组蛋白表达情况。蛋白质印迹（Western blotting）分析重组蛋白对6 × His标签单克隆抗体、感染广州管圆线虫的小鼠和患者血清的免疫反应性。结果 AcCBL1和AcCBL2基因PCR扩增产物分别约为1 000、1 100 bp。SDS-PAGE结果显示,rAcCBL1为可溶表达蛋白,相对分子质量（M_r）约为37 800;rAcCBL2主要以包涵体形式存在,约为M_r 40 800。Western blotting结果显示,rAcCBL1和rAcCBL2能与6 × His标签单克隆抗体、感染广州管圆线虫的小鼠和患者血清特异性结合。结论成功克隆和表达AcCBL1和AcCBL2基因,二者表达产物均具有良好的免疫反应性。

关键词: 广州管圆线虫, 半胱氨酸蛋白酶, 表达, 鉴定

Abstract:

Objective To clone and express the cysteine protease genes AcCBL1 and AcCBL2 of Angiostrongylus cantonensis, and analyze the immunoreactivity of the purified recombinant proteins. Methods Two cysteine protease genes AcCBL1 and AcCBL2 were identified from Angiostrongylus cantonensis fourth-stage larvae cDNA library, and cloned into pET-28a vector to generate recombinant plasmids pET-28a-AcCBL1 and pET-28a-AcCBL2, respectively. The constructed recombinant plasmids were transformed into（E. coli） BL21（DE3） respectively for expression under the induction of isopropyl β-D-1-thiogalactopyranoside(IPTG). The recombinant proteins rAcCBL1 and rAcCBL2 were purified by Ni-NTA affinity chromatography and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The immunoreactivity of rAcCBL1 and rAcCBL2 were analyzed by Western blotting with 6 × His tag antibody, as well as anti-sera from mice infected with A. cantonensis and patients. Results PCR generated a specific band at 1 000 bp and 1 100 bp for AcCBL1 and AcCBL2, respectively, consistent with the theoretical values. The constructed vector pET-28a-AcCBL1 and pET-28a-AcCBL2 were confirmed by gene sequencing. SDS-PAGE revealed that rAcCBL1 was soluble with relative molecular mass of 37 800, and rAcCBL2 was expressed as inclusion body in E. coli with relative molecular mass of 40 800. Western blotting showed that the two purified recombinant proteins can be recognized by 6 × His tag antibody, as well as anti-sera from mice infected with A. cantonensis and patients. Conclusion AcCBL1 and AcCBL2 genes have been expressed in prokaryotic expression system. The two recombinant proteins both show adequate immunoreactivity.

Key words: Angiostrongylus cantonensis, Cysteine protease, Expression, Identification

中图分类号:

R383.19

白慧芳, 柯琪文, 陈韵秋, 刘芷晴, 张玲敏, 吴春云, 袁桂秀, 詹希美, 程梅. 广州管圆线虫半胱氨酸蛋白酶AcCBL1和AcCBL2基因的克隆、表达及鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(5): 469-473.

Hui-fang BAI, Qi-wen KE, Yun-qiu CHEN, Zhi-qing LIU, Ling-min ZHANG, Chun-yun WU, Gui-xiu YUAN, Xi-mei ZHAN, Mei CHENG. Cloning, expression and identification of cysteine protease genes AcCBL1 and AcCBL2 of Angiostrongylus cantonensis[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2018, 36(5): 469-473.

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