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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。
Lectins belong to the β-galactoside binding protein family and exist widely in insects and play an important role in the regulation of immunity. Studying lectins in Aedes aegypti has important theoretical significance. In this study, primers were designed according to the sequence of galectin12 gene from Aedes aegypti. The galectin12 fragment was amplified by q RT-PCR and cloned into pMD-18T cloning vector. The positive clones were selected for digestion and Sequencing and verification, the target gene fragment was ligated to the pET-32a vector for prokaryotic expression according to the restriction site to obtain a 23 kD protein of interest, and the target protein was purified by using His enzyme affinity chromatography technology to obtain the purified protein, The results of bioassay showed that Galectin 12 could inhibit the toxicity of Bt to mosquitoes. The results provide a molecular basis for further verifying the mechanism of agglutinin participating in Aedes aegypti resistance to Bt Cry toxin.