恶性疟原虫环子孢子蛋白单链抗体与按蚊cecropin A融合基因的构建、原核表达及生物学活性的初步分析

CONSTRUCTION, PROKARYOTIC EXPRESSION AND PRELIMINARY IDENTIFICATION OF THE BIOACTIVITY OF THE FUSION GENE M2A10 AND CECROPIN A

根据按蚊偏嗜性密码子对鼠源性恶性疟原虫环子孢子蛋白单链抗体2a10基因进行改造,并融合按蚊抗菌肽cecropin A编码基因。将获得的cecrop-m2a10融合基因克隆入原核表达载体pET32a(+)中,对表达产物进行SDS-PAGE、Western-blot分析并利用琼脂糖扩散法检测其抗菌活性。结果对鼠源性环子孢子蛋白单链抗体2a10中6种氨基酸的170个核苷酸进行了改造,融合cecropinA编码基因,成功构建了cecrop-m2a10融合基因。靶基因在大肠杆菌中以融合蛋白和包涵体的形式高效表达,包涵体经尿素溶解变性及透析复性后表达蛋白的纯度达75%并具备抗大肠杆菌DH5α的活性。本实验为进一步研究cecrop-m2a10在转基因蚊中的多重抗病原效应提供了基础。

The coding sequence of a single chain antibody （2a10） against circumsporozoite protein （CSP） of Plasmodium falciparum was modified according to Anopheles gambiae preferred codons. Totally, 170 nucleotides in six kinds of amino acids were replaced and then linked with the coding sequence from A. gambiae cecropin A. Subsequently, the fusion gene, cecrop-m2a10, was subcloned into the expression vector pET32a （ ＋ ） and expressed in E. coli BL21 （DE3）. Expression products were analyzed by SDS-PAGE, Western-blot and Agar-diffusion method. Results indicated that the cecrop-m2a10 fusion gene was correctly cloned into expression vector pET32a （ ＋ ） and was highly expressed in E. coli as an insoluble form. After the collection of inclusion body, denaturing lysis and refoding dialysis, the purity of the expression product was over 70% and showed bacteriostasis inhibition to the growth of E. coli DHSa. Our experiment provided significant data for-further study on the multiple anti-pathogen effect of cecrop-m2a10 gene in transgenic mosqitoes.