[1]陈萌萌,潘渊婷,范志宇,等.兔源抗凋亡蛋白Bcl-2的表达、抗体的制备及初步应用[J].江苏农业学报,2019,(06):1397-1401.[doi:doi:10.3969/j.issn.1000-4440.2019.06.018]
　CHEN Meng-meng,PAN Yuan-ting,FAN Zhi-yu,et al.Prokaryotic expression, polyclonal antibody preparation and preliminary application of rabbit anti-apoptotic protein Bcl-2[J].,2019,(06):1397-1401.[doi:doi:10.3969/j.issn.1000-4440.2019.06.018]
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兔源抗凋亡蛋白Bcl-2的表达、抗体的制备及初步应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2019年06期
页码:
1397-1401
栏目:
畜牧兽医·水产养殖
出版日期:
2019-12-31

文章信息/Info

Title:
Prokaryotic expression, polyclonal antibody preparation and preliminary application of rabbit anti-apoptotic protein Bcl-2
作者:
陈萌萌; 潘渊婷; 范志宇; 胡波; 宋艳华; 魏后军; 仇汝龙; 朱伟峰; 徐为中; 王芳
(江苏省农业科学院兽医研究所/农业部动物疫病诊断与免疫重点开放实验室/国家兽用生物制品工程技术研究中心，江苏南京210014)
Author(s):
CHEN Meng-meng; PAN Yuan-ting; FAN Zhi-yu; HU Bo; SONG Yan-hua; WEI Hou-jun; QIU Ru-long; ZHU Wei-feng; XU Wei-zhong; WANG Fang
（Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China）
关键词:
兔源抗凋亡蛋白Bcl-2; 原核表达; 亲和层析; 多抗制备
Keywords:
rabbit anti-apoptotic protein Bcl-2; prokaryotic expression; affinity chromatography; preparation of polyclonal antibodies
分类号:
S858.291
DOI:
doi:10.3969/j.issn.1000-4440.2019.06.018
文献标志码:
A
摘要:
为了原核表达兔源抗凋亡蛋白Bcl-2，并诱导表达纯化该蛋白后制备多抗，采用RT-PCR扩增Bcl-2编码序列，并将该序列克隆至pET-32a(+)载体，获得重组pET-32a-Bcl-2质粒转化BL21(DE3)，筛选最佳表达条件，通过亲和层析纯化目的蛋白质，最后经Western blot鉴定后免疫小鼠。结果显示，成功扩增Bcl-2编码序列并构建了pET-32a-Bcl-2表达载体；SDS-PAGE结果显示，在16 ℃，5 h，0.5 mmol/L IPTG的表达条件下，重组蛋白Bcl-2能够高效可溶性表达；Western blot结果表明纯化后的表达产物为高纯度的Bcl-2重组蛋白，将该蛋白质免疫小鼠后获得了特异性抗体，该抗体能够特异性识别重组Bcl-2蛋白，并应用该抗体鉴定RK13-B细胞中Bcl-2蛋白的过表达。
Abstract:
In order to construct the prokaryotic expression system of Bcl-2, induce expression of Bcl-2 fusion protein, prepare polyclonal antibodies and conduct preliminary application, the coding sequence of Bcl-2 was amplified by RT-PCR and cloned into the pET-32a (+) vector. The pET-32a-Bcl-2 was transformed to BL21(DE3), and the optimal expression conditions were determined. The target protein was purified by affinity chromatography and identified by Western blot. The results showed that Bcl-2 coding sequence was successfully amplified, and its expression vector was constructed. The results of SDS-PAGE showed that the recombinant protein Bcl-2 was highly soluble under optimization condition (16 ℃, 5 h, 0.5 mmol/L IPTG). The results of Western blot indicated that the purified product was recombinant protein Bcl-2 with higher purity, and the specific antibody from mice immunized with recombinant protein Bcl-2. In addition, the antibody could specifically recognize the recombinant protein Bcl-2, and identify the overexpression of Bcl-2 protein in RK13-B cells.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期：2019-05-17 基金项目：国家自然科学基金项目(31702274)；现代农业产业技术体系建设专项基金项目（CARS-43-C-1） 作者简介：陈萌萌 (1987-) ，女，江苏连云港人，博士，助理研究员，主要从事家兔疾病防治与兽医生物技术研究。(E-mail)moonchen2010@yeah.net 通讯作者：王芳，(E-mail)rwangfang@126.com
更新日期/Last Update: 2020-01-09