埃及伊蚊galectin12基因的克隆、表达及杀虫活性测定

凝集素属于β-半乳糖苷结合蛋白家族,广泛存在于昆虫的体内,对其免疫调控起重要作用,研究埃及伊蚊（Aedes aegypti）中的凝集素具有重要理论意义。本研究以埃及伊蚊凝集素galectin12基因序列设计引物,通过q RT-PCR扩增获得埃及伊蚊凝集素galectin12基因片段,将其连接到p MD-18T克隆载体,筛选阳性克隆子进行酶切以及测序验证,根据酶切位点将目的基因片段连接到p ET-32a载体进行原核表达,获得23 k D的目的蛋白,并采用His酶亲和层析技术对目的蛋白进行纯化,获得纯化蛋白,并进行生物活性测定,结果表明,Galectin12能够抑制Bt对蚊虫的毒力。研究结果为进一...

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Published in农业生物技术学报 Vol. 23; no. 11; pp. 1472 - 1477
Main Author 胡小华 黄鲜晖 刘健楠 陈倩倩 孙振裕 关雄 张灵玲
Format Journal Article
LanguageChinese
Published 2015
Subjects
galectin12
克隆
埃及伊蚊
表达
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Summary:凝集素属于β-半乳糖苷结合蛋白家族,广泛存在于昆虫的体内,对其免疫调控起重要作用,研究埃及伊蚊（Aedes aegypti）中的凝集素具有重要理论意义。本研究以埃及伊蚊凝集素galectin12基因序列设计引物,通过q RT-PCR扩增获得埃及伊蚊凝集素galectin12基因片段,将其连接到p MD-18T克隆载体,筛选阳性克隆子进行酶切以及测序验证,根据酶切位点将目的基因片段连接到p ET-32a载体进行原核表达,获得23 k D的目的蛋白,并采用His酶亲和层析技术对目的蛋白进行纯化,获得纯化蛋白,并进行生物活性测定,结果表明,Galectin12能够抑制Bt对蚊虫的毒力。研究结果为进一步验证凝集素参与埃及伊蚊抵御Bt Cry毒素过程的机理奠定分子基础。
Bibliography:Aedes aegypti; Cloning; Expression; galectin12
Galectins are a family of β- galactoside- binding proteins that widely distribute among insect species which play an important role in insect immune regulation. Thus the research of Aedes aegypti galectins is of great significance. In this study, primers were designed based on the A. aegypti galectin12 gene sequence,and the entire coding region of galectin12 gene was amplified by q RT- PCR with extracted total RNA. PCR product was purified and ligated to p MD- 18 T cloning vector. The selected positive clone was used for digested analysis and nucleotide sequencing. After endonucleases digestion, fragments were connected to p ET-32 a vector and obtained 23 k D protein. In addition, the expressed protein was purified by Ni- NTA affinity chromatography, and obtained purified protein bioassay result indicated that galectin12 could supporess mosquito Bt toxies. Cloning, expression and purification of Galectin 12 would help to furtherly understand the mechanism of galec
ISSN:1674-7968