鸡α-2,3-唾液酸转移酶Ⅰ基因的克隆和真核表达载体的构建

›› 2013, Vol. 40 ›› Issue (1): 21-24.

鸡α-2,3-唾液酸转移酶Ⅰ基因的克隆和真核表达载体的构建

刘媛媛, 王俊亚, 张晓娟, 陈礼朋, 岳旭龙, 高文明, 李双亮, 崔保安, 李新生

河南农业大学牧医工程学院, 河南郑州 450002

收稿日期:2012-07-26 出版日期:2013-01-20 发布日期:2013-01-14
通讯作者: 李新生(1969-),河南人,博士,副教授,主要从事分子病毒学研究。E-mail:harmony69@gmail.com;Tel:0371-63558519 E-mail:harmony69@gmail.com
作者简介:刘媛媛(1987-),女,河南人,硕士,研究方向:分子病毒学。
基金资助:
河南省科技成果转化项目"禽类重大疫病多联疫苗的中试与转化"(122201110027)。

Cloning and Construction of Eukaryotic Expression Vector of Chicken ST3GAL Ⅰ Gene

LIU Yuan-yuan, WANG Jun-ya, ZHANG Xiao-juan, CHEN Li-peng, YUE Xu-long, GAO Wen-ming, LI Shuang-liang, CUI Bao-an, LI Xin-sheng

College of Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China

Received:2012-07-26 Online:2013-01-20 Published:2013-01-14

摘要/Abstract

摘要： 试验利用Trizol法从鸡肠道组织提取总RNA,采用特异性引物通过RT-PCR扩增出α-2,3-唾液酸转移酶Ⅰ(α-2,3-sialyltransferaseⅠ,ST3GALⅠ)基因的cDNA片段,并将其克隆至pGEM-T easy载体,获得阳性重组质粒,再以阳性重组质粒为模板亚克隆ST3GALⅠ基因的完整ORF区,定向插入到真核表达载体pcDNA3.1(+)上,进行PCR、限制性酶切和DNA序列分析鉴定。结果表明,ST3GALⅠ基因全长1029 bp,测序结果同GenBank数据库收录的序列一致,无任何密码子缺失与突变,插入到真核表达载体pcDNA3.1(+)上的目的基因大小方向均正确。本研究成功构建了pcDNA3.1(+)-ST3GALⅠ真核表达载体,为下一步的真核表达及对ST3GALⅠ基因的功能研究奠定了基础。

关键词: 禽流感病毒; 受体; α-2,3-唾液酸转移酶Ⅰ; 真核表达载体

Abstract: Total RNA was extracted from intestinal of chicken by the method of Trizol. The α-2, 3-sialyltransferase Ⅰ(ST3GAL Ⅰ) gene’s cDNA was amplified by reverse transcription polymerase chain reaction with specific primer. The amplified fragment was cloned into pGEM-T easy vector, then got the recombinant plasmid. Sub-cloning the ORF of ST3GAL Ⅰ gene of pGEM-ST3GAL Ⅰ and inserted the eukaryotic expression vector pcDNA3.1(+). The target segment in recombinant plasmid was confirmed by PCR, restriction enzyme digestion and sequencing methods. The results indicated that ST3GAL Ⅰ gene was 1029 bp in length, the result of sequence was consistent with the sequence which included in GenBank, and without any mutation and deletion of codon. The length and directions of gene which inserted to eukaryotic expression vector pcDNA3.1 (+) were all correct. The recombinant eukaryotic expression vector pcDNA3.1(+)-ST3GAL Ⅰ had been constructed successfully, which laid the foundation for further eukaryotic expressing and studying of functional of ST3GAL Ⅰ gene.

Key words: avain influenza virus; receptor; α-2,3-sialyltransferase Ⅰ; eukaryotic expression vector

中图分类号:

刘媛媛, 王俊亚, 张晓娟, 陈礼朋, 岳旭龙, 高文明, 李双亮, 崔保安, 李新生. 鸡α-2,3-唾液酸转移酶Ⅰ基因的克隆和真核表达载体的构建[J]. , 2013, 40(1): 21-24.

LIU Yuan-yuan, WANG Jun-ya, ZHANG Xiao-juan, CHEN Li-peng, YUE Xu-long, GAO Wen-ming, LI Shuang-liang, CUI Bao-an, LI Xin-sheng. Cloning and Construction of Eukaryotic Expression Vector of Chicken ST3GAL Ⅰ Gene[J]. , 2013, 40(1): 21-24.

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