硒蛋白<em>Sep15</em>基因克隆、定点突变及其原核表达

中国生物工程杂志

2011, Vol. 31

Issue (8): 12-17

研究报告

硒蛋白Sep15基因克隆、定点突变及其原核表达

周继昌^1,2, 李黛淋², 汤加勇², 赵华², 刘小立¹, 朱玉梅¹, 徐健¹

1. 深圳市慢性病防治中心分子生物学实验室深圳 518020;
2. 四川农业大学动物营养研究所成都 611134

Gene Cloning,Site-directed Mutagenesis and Prokaryotic Expression of Porcine Sep15

ZHOU Ji-chang^1,2, LI Dai-lin², TANG Jia-yong², ZHAO Hua², LIU Xiao-li¹, ZHU Yu-mei¹, XU Jian¹

1. Molecular Biology Lab,Shenzhen Center of Chronic Disease Control,Shenzhen 518020,China;
2. Institute of Animal Science,Sichuan Agricultural University,Chengdu 611134,China

全文: PDF(1741 KB) HTML

摘要：

克隆鉴定猪硒蛋白Sep15基因( Sep15 ),将其突变实现原核表达,为以猪为模型研究Sep15功能奠定基础。实验以RT-PCR从猪脾总RNA扩增出含开放阅读框(ORF)至poly(A)共1230 bp的 Sep15 cDNA,3'-非翻译区Sec插入元件为2型,489 bp的ORF及对应氨基酸序列与人相应序列的相似度分别为85.1%和92.7%,ORF含一个硒代半胱氨酸(Sec)密码子TGA,定点突变为半胱氨酸(Cys)的TGC后,经载体pET30转入大肠杆菌BL21(DE3),0.4 mmol/L IPTG诱导表达3 h获得融合表达产物;该产物在Western blot检测中与人Sep15 Sec下游肽段的商品化多抗产生特异性免疫印迹。猪 Sep15 被首次成功克隆并鉴定,其Cys突变体的原核表达产物与人Sep15 C-端抗体存在交叉免疫反应。

关键词： Sep15; 定点突变; 原核表达; 猪

Abstract:

To clone and identify the gene of porcine selenoprotein 15kDa(Sep15), Sep15 ,conduct the site-directed mutagenesis and prokaryotic expression for the further functional study of Sep15 using pig models. The total RNA was extracted from pig spleen for RT-PCR,and then the Sep15 cDNA sequence of 1230 bp containing the open reading frame(ORF)till to poly(A)tail was cloned. The 489 bp of ORF sequence had 85.1% identity to that of human,while their amino acid sequences had 92.7% identity. Like those in various mammals,the porcine Sep15 cDNA bore the common characteristics of an in-frame TGA for selenocysteine(Sec)and a Sec insertion sequence element of Form 2 located in the 3'-untranslated region. By inverse PCR,the TGA codon for Sec was mutated into TGC for cysteine(Cys). The mutant was recombined to pET30 vector and expressed in E. coli BL21(DE3)under the induction of 0.4 mmol/L IPTG for 3h. The expressed recombinant protein with His-tag detected by SDS-PAGE was about 23kDa. In Western blot assays,both the rabbit anti-sera generated by the purified recombinant protein and the commercial murine antibody against the peptide downstream the Sec residue of human Sep15 were able to detect the recombinant protein. In conclusion,the porcine Sep15 was cloned and identified for the first time,and its molecular characteristics shared high similarity with those of human SEP15 . And the Cys-mutant of porcine Sep15 cross reacted with the antibody against the C-terminal of human Sep15.

Key words: Sep15 Site-directed mutagenesis Prokaryotic expression Pig

收稿日期: 2010-11-12 出版日期: 2011-08-25

ZTFLH:

Q786

基金资助:

中国博士后科学基金(20070410391)、广东省医学科研基金(A2009607) 资助项目

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引用本文:

周继昌, 李黛淋, 汤加勇, 赵华, 刘小立, 朱玉梅, 徐健. 硒蛋白Sep15基因克隆、定点突变及其原核表达[J]. 中国生物工程杂志, 2011, 31(8): 12-17.

ZHOU Ji-chang, LI Dai-lin, TANG Jia-yong, ZHAO Hua, LIU Xiao-li, ZHU Yu-mei, XU Jian. Gene Cloning,Site-directed Mutagenesis and Prokaryotic Expression of Porcine Sep15. China Biotechnology, 2011, 31(8): 12-17.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I8/12

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