淡色库蚊视蛋白主要抗原域的原核表达及多克隆抗体制备  

Prokaryotic expression of the major antigenic domain in opsin from Culex pipiens pallens and preparation of polyclonal antibodies

作　　者：邹萍[1] 孙艳[1] 孙海波[1] 朱昌亮[1] 

机构地区：[1]南京医科大学病原生物学系,江苏南京210029

出　　处：《中国病原生物学杂志》2012年第5期363-366,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金青年科学基金项目(No.30901244)

摘　　要：目的为进一步研究蚊视蛋白参与杀虫剂抗性的相关分子机制和将其作为现场抗性检测靶标提供多克隆抗体。方法以前期制备的重组质粒pGEM-T Easy/opsin为模板,通过基因工程手段克隆视蛋白基因胞外区片段,构建原核载体,IPTG诱导表达蛋白,并利用His亲和层析以及肠激酶酶切片段获得纯度较高的蛋白,免疫新西兰大白兔,制备多克隆抗体,Western blot鉴定抗体特异性。结果成功构建淡色库蚊视蛋白主要抗原域的原核表达载体,经IPTG 1mmol/L诱导3h后高效表达可溶性重组蛋白,经His亲和层析及肠激酶酶切后获得纯度较高的蛋白,制备的多克隆抗体经Western blot证实能特异性地识别蚊视蛋白而不与非特异性蛋白结合。结论成功构建了淡色库蚊视蛋白主要抗原域的原核表达载体,制备的抗重组蛋白多克隆抗体特异性好,效价较高,为进一步研究视蛋白参与杀虫剂抗性的分子机制和将其作为现场抗性检测靶标提供了基础。Objective To investigate how opsin from Culex pipiens pallens participates in insecticide resistance and to provide a specific rabbit polyclonal antibody against opsin to test for resistance.Methods The sequence for a main antigenic domain in the opsin gene of Culex pipiens pallens was amplified by PCR from a pGEM-T Easy/opsin recombinant plasmid prepared ahead of time and was inserted into an inducible prokaryotic expression plasmid,pET-32a（＋）.Expression of Opsin-His fusion protein was induced with IPTG and purified on a Ni＋ affinity column followed by cleavage with enterokinase.The purified opsin protein was used to immunize New Zealand rabbits to prepare polyclonal antibodies with specificity as defined by Western blotting.Results A recombinant vector of the major antigenic domain of Culex pipiens pallens opsin was successfully constructed.The fusion protein was abundantly expressed 3 hours after the recombinant vectors were induced with 1 mmol/L IPTG.Western blot analysis indicated that the opsin-expressing protein was specifically detected by antibodies against Culex pipiens pallens.Conclusion A recombinant vector of the main antigenic domain of Culex pipiens pallens opsin was successfully constructed.Polyclonal antibodies that were prepared had a high titer and specificity and should further the study of how protein opsin participates in insecticide resistance.

关 键 词：淡色库蚊 抗药性 视蛋白 原核表达 多克隆抗体 

分 类 号：R384.1[医药卫生—医学寄生虫学]