二斑叶螨几丁质酶基因的原核表达及多克隆抗体制备

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...

Full description

Saved in:
Bibliographic Details
Published in东北农业大学学报 Vol. 46; no. 1; pp. 62 - 67
Main Author 张道伟 陈静 张正玲 曾燕玲 郭玉双
Format Journal Article
LanguageChinese
Published 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%遵义医学院生化与分子生物学教研室,贵州 遵义,563009%贵州省烟草科学研究院,贵阳,550083 2015
Subjects
二斑叶螨
几丁质酶
原核表达
多克隆抗体
几丁质酶
chitinase
Tetranychus urticae
原核表达
polyclonal antibody
二斑叶螨
prokaryotic expression
多克隆抗体
Online AccessGet full text
ISSN1005-9369

Cover

More Information
Summary: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。
Bibliography:ZHANG Daowei;CHEN Jing;ZHANG Zhengling;ZENG Yanling;GUO Yushuang （1. School of Life Sciences Zunyi Normal College/Key Laboratory of Protection and Utilization of Animal Resource in Chishui River Basin, Zunyi Guizhou 563002, China 2. Department of Biochemistry and Molecular Biology, Zunyi Medical College, Zunyi Guizhou 563009 China; 3. Guizhou Tobacco Research Institute, Guiyang 550083, China）
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 specifi
ISSN:1005-9369