[1]周智敏,韩崇选*. 草兔卵透明带蛋白2的克隆与原核表达[J].西北林学院学报,2016,31(2):181-187.[doi:10.3969/j.issn.1001-7461.2016.02.31]
　ZHOU Zhi-min,HAN Chong-xuan*. Gene Cloning and Prokaryotic Expression of Lepus Capensis Zona Pellucida Protein 2[J].JOURNAL OF NORTHWEST FORESTRY UNIVERSITY,2016,31(2):181-187.[doi:10.3969/j.issn.1001-7461.2016.02.31]
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 草兔卵透明带蛋白2的克隆与原核表达()
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《西北林学院学报》[ISSN:1001-7461/CN:61-1202/S]

卷:
第31卷
期数:
2016年第2期
页码:
181-187
栏目:
出版日期:
2016-03-31

文章信息/Info

Title:
 Gene Cloning and Prokaryotic Expression of Lepus Capensis Zona Pellucida Protein 2
文章编号:
1001-7461(2016)02-0181-07
作者:
 周智敏; 韩崇选*
 （西北农林科技大学 林学院，陕西 杨陵 712100）
Author(s):
 ZHOU Zhi-min; HAN Chong-xuan*
 （College of Forestry，Northwest A&F University，Yangling，Shaanxi 712100，China）
关键词:
 ; 卵透明带; 草兔; 抗原表位; ZP2; 诱导表达
Keywords:
 ; zona pellucida;  Lepus Capensis;  epitope;  ZP2;  optimum expression
分类号:
S764.5
DOI:
10.3969/j.issn.1001-7461.2016.02.31
文献标志码:
A
摘要:
 哺乳动物卵透明带蛋白具有极强抗原性，产生的抗卵透明带蛋白抗体可以有效阻碍精卵结合。为了探究草兔卵透明带2 (zona pellucida 2，ZP2)蛋白抗原性，以及定位抗原表位。根据GenBank上欧洲兔的卵透明带2基因序列，设计5′端磷酸化的反转录引物用于合成特异cDNA作为模板，通过普通PCR扩增草兔ZP2基因。测序获得的2 184 bp序列比对成功后，预测其信号肽和跨膜区域分别位于ZP235-56和ZP2698-717。将克隆获得954 bp的hZP2基因连入pET-28a(+)的EcoRⅠ和XhoⅠ酶切位点之间，成功构建了重组原核表达载体pET-28a-hZP2后转化大肠杆菌（Escherichia coli，E.coli）BL21。重组的E.coli BL21经诱导后表达41.17 kD可溶性的hZP2融合蛋白，经优化诱导表达体系后，确定较好的诱导条件是37℃下 0.7 mmol·L-1 IPTG诱导15 h。经Western blot鉴定，结果表明相对于表达ZP2大片段基因，hZP2融合蛋白表达量明显提高。
Abstract:
 Mammalian zona pellucida (ZP) has great antigenicity.After inoculating，the antibody of ZP can effectively hinder sperm-egg interaction.To understand more about the antigenicity of Lepus Capensis ZP2 and map its epitope，we designed a special reverse transcription primer，in which 5′ ends were phosphorylated，based on ZP2 cDNA sequence of Oryctolagus cuniculus.The first strand of cDNA was synthesized and then acted as template to amplify the open reading frame of Lepus capensis ZP2 gene.ZP2 cDNA contained 2 184 nucleotides，its signal peptide and transmembrane helices was located at ZP235-56 and ZP2698-717,respectively.The hZP2 cDNA was amplified and contained 954 nucleotides.After inserting the hZP2 cDNA into pET-28a(+) plasmids，the recombinant pET-28a-ZP2 vector was then transformed into Escherichia coli BL21 competent cells.Temperature，IPTG concentration and time were optimized to increase the level of expression of hZP2 gene.The recombinant E.coli BL21 was induced and expressed dissoluble hZP2 fusion proteins，which molecular weight were 41.17 kD.Preferably,recombinant E.coli BL21 grew at 37℃ and was induced 15 h using 0.7 mmol·L-1 IPTG and then expressed more hZP2 fused proteins.The level of expression of hZP2 fusion proteins was much more than expression of ZP2f fusion proteins.

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备注/Memo

备注/Memo:
 收稿日期：2015-04-09修回日期：2015-05-14
基金项目：国家林业局林业公益性行业科研专项（201404405）。
作者简介：周智敏，男，在读硕士，研究方向：草兔数量的免疫不育调控。E-mail：zhouzhimin6@gmail.com
*通信作者：韩崇选，男，研究员，教授，研究方向：有害脊椎动物调控。E-mail：sendakingcat@nwsuaf.edu.cn
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