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2025年8月3日 星期日
农业生物技术学报  2019, Vol. 27 Issue (11): 2083-2090    DOI: 10.3969/j.issn.1674-7968.2019.11.019
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
猪P58IPK蛋白多克隆抗体的制备
卓严玲1, 吕其壮1, 2, *, 邓家华1, 梁小梅1, 梁小妹1, 覃婷1, 张柱青1
1 玉林师范学院 生物与制药学院,玉林 537000;
2 广西农产资源化学与生物技术重点实验室,玉林 537000
Preparation of Polyclonal Antibody Against Porcine (Sus scrofa) P58IPK Protein
ZHUO Yan-Ling1, LV Qi-Zhuang1, 2, *, DENG Jia-Hua1, LIANG Xiao-Mei1, LIANG Xiao-Mei1, QIN Ting1, ZHANG Zhu-Qing1
1 College of Biology & Pharmacy, Yulin Normal University, Yulin 537000, China;
2 Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, Yulin 537000, China
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摘要 P58IPK (58 kD inhibitor of PKR)作为宿主细胞分子伴侣蛋白,在调节机体抗病毒反应和免疫应答方面发挥着重要作用。本研究拟克隆猪(Sus scrofa) P58IPK基因CDS序列并构建其原核表达载体,纯化P58IPK蛋白并以此为抗原制备多克隆抗体,以期为后续研究猪圆环病毒2型(Porcine circovirus type 2, PCV2)与宿主之间的互作关系提供基础材料。根据猪P58IPK全基因序列(GenBank No. HQ287801.1)设计引物,以反转录PCR (reverse transcription PCR, RT-PCR)方法扩增P58IPK基因CDS序列(去除信号肽序列),经NdeⅠ/XbaⅠ双酶切后定向克隆至原核表达载体pCzn1;将其转化Arctic-ExpressTM大肠杆菌(Escherichia coli)进行诱导表达;表达产物经变性、复性及His-Band Ni+层析柱亲和纯化,将纯化产物His-P58IPK重组蛋白作为抗原免疫兔子(Oryctolagus?cuniculus)制备多克隆抗体。抗体纯化后,分别利用间接ELISA和Western blot方法对其效价和特异性进行检测。结果显示,克隆所得的猪P58IPK基因CDS序列长度为1 425 bp,表达产物His-P58IPK重组蛋白主要以包涵体形式存在,分子量约为54.44 kD;由此蛋白制备的多抗效价在1∶512 000以上,能够特异性识别目标蛋白(包括重组蛋白His-P58IPK和猪P58IPK蛋白)。本研究成功克隆了猪P58IPK多克隆抗体,可为深入研究猪P58IPK蛋白功能和PCV2病毒与宿主之间的互作机制提供基础资料。
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卓严玲
吕其壮
邓家华
梁小梅
梁小妹
覃婷
张柱青
关键词 猪P58IPK蛋白原核表达与纯化多克隆抗体    
Abstract:As a host cell chaperone protein, P58IPK (58 kD inhibitor of PKR) plays an important role in regulating the body's antiviral and immune response. In this study, CDS region of porcine (Sus scrofa) P58IPK gene was cloned and its prokaryotic expression vector was constructed. Purified P58IPK protein was used as an antigen to produce polyclonal antibody, which would provide basic materials for the further study on the interplay between Porcine circovirus type 2 (PCV2) and its host. A specific pair of primers with the usage of amplifying the partial P58IPK CDS sequence (containing no signal peptide sequence) by reverse transcription PCR (RT-PCR) was designed according to the complete genome sequence of porcine P58IPK (GenBank No. HQ287801.1). After double digestion by NdeⅠ/XbaⅠ restriction enzymes, the products were cloned into the prokaryotic expression vector pCzn1. After sequencing, the generated recombinant expression plasmid pCzn1-P58IPK was transformed into bacterium Arctic ExpressTM Escherichia coli. The expressed His-P58IPK recombinant proteins were denaturated, renaturated and purified by using His-Band Ni+ affinity chromatography, then were used as target antigens to immunize rabbits (Oryctolagus?cuniculus) to prepare polyclonal antibody (pAb). After the pAb was purified, its titer and specificity were detected by indirect ELISA and Western blot, respectively. The results showed that the length of the cloned P58IPK gene fragment was 1 425 bp, the expressed His-P58IPK recombinant protein with a molecular weight of about 54.44 kD mainly existed in a form of inclusion body. The titer of the pAb prepared from this recombinant protein was above 1∶512 000, and the specificity of this pAb was verified very well as it could not only recognize the His-P58IPK recombinant protein but also porcine P58IPK protein. These data indicated that the partial CDS region of porcine P58IPK gene has been successfully cloned, expressed, purified and the prepared polyclonal antibody could be used for the detection of porcine P58IPK protein. The obtained polyclonal antibody could be used as basic material for further studying of the function of porcine P58IPK protein and interaction mechanism between PCV2 virus and the host.
Key wordsPorcine P58IPK protein    Prokaryotic expression and purification    Polyclonal antibody
收稿日期: 2019-04-19     
ZTFLH:  S852.65+9.2  
  Q785  
基金资助:国家自然科学基金(No. 31860708)和广西自然科学基金(No. 2017GXNSFBA198025)
通讯作者: lvqizhuang062@163.com   
引用本文:   
卓严玲, 吕其壮, 邓家华, 梁小梅, 梁小妹, 覃婷, 张柱青. 猪P58IPK蛋白多克隆抗体的制备[J]. 农业生物技术学报, 2019, 27(11): 2083-2090.
ZHUO Yan-Ling, LV Qi-Zhuang, DENG Jia-Hua, LIANG Xiao-Mei, LIANG Xiao-Mei, QIN Ting, ZHANG Zhu-Qing. Preparation of Polyclonal Antibody Against Porcine (Sus scrofa) P58IPK Protein. 农业生物技术学报, 2019, 27(11): 2083-2090.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.11.019     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I11/2083
 
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