二斑叶螨几丁质酶基因的原核表达及多克隆抗体制备  

Expression and polyclonal antibody preparation of chitinase gene in Tetranychus urticae Koch

作　　者：张道伟[1] 陈静[2] 张正玲[1] 曾燕玲[1] 郭玉双 

机构地区：[1]遵义师范学院生命科学学院,赤水河流域动物资源保护与应用研究重点实验室,贵州遵义563002 [2]遵义医学院生化与分子生物学教研室,贵州遵义563009 [3]贵州省烟草科学研究院,贵阳550083

出　　处：《东北农业大学学报》2015年第1期62-67,共6页Journal of Northeast Agricultural University

基　　金：贵州省科技厅项目(黔科合J字[2013]2307号);遵义师范学院博士基金(BSJJ201206)

摘　　要：以制备的二斑叶螨c DNA为模板,克隆二斑叶螨几丁质酶Se Chi基因功能结构域催化区片段,大小为786 bp。将该序列片段克隆到表达载体p ET-32a(+)中,获得多克隆原核表达载体p ET-32a-Tu Chi。重组质粒经酶切测序鉴定后转化大肠杆菌BL21(DE3),经0.6 mmol·L-1IPTG诱导4 h后高效表达约45 ku可溶性重组蛋白;经His亲和层析洗脱和浓缩获得高纯度的重组蛋白免疫新西兰大白兔制备Tu Chi多克隆抗体。制备的多克隆抗体经Western Blot证实能特异性地识别几丁质酶而不与非特异性蛋白结合。ELISA分析表明制备的抗体效价达1??80 000,效价较高,为进一步研究二斑叶螨几丁质酶相关功能奠定基础。The sequence of part chitinase gene of Tetranychus urticae was amplified by PCR from c DNA. The sequence length of this domain was 786 bp. Then, the gene was cloned into p ET- 32a（＋）prokaryotic expressive vector, and the constructed recombinant plasmids p ET- 32a- Tu Chi was transformed into the host bacteria E. coli BL21（DE3）. About 45 ku fusion protein was abundantly expressed at 4 h after the recombinant vector was induced with 0.6 mmol· L-1IPTG. The fusion protein was purified on a Ni＋affinity column. The purified chitinase protein was used to immunize New Zealand rabbits for preparing polyclonal antibody with specificity as defined by Western Blot. ELISA analysis showed that the titer of the polyclonal antibody was 1-80 000, polyclonal antibody that was prepared had a high titer and specificity, and the results laid the foundation to further study the function of the chitinase in Tetranychus urticae.

关 键 词：二斑叶螨 几丁质酶 原核表达 多克隆抗体 

分 类 号：Q786[生物学—分子生物学]