中国人乳β-酪蛋白基因克隆表达及生物信息学分析  

Cloning, Expression and Bioinformatic Analyses of Chinese Human β-casein Gene

作　　者：任皓威[1,2,3] 李春[2,3] 刘宁[1,2,3] 顾鑫[1,2,3] 

机构地区：[1]国家乳业工程技术研究中心黑龙江省乳品工业技术开发中心,哈尔滨150028 [2]乳品科学教育部重点实验室东北农业大学食品学院,哈尔滨150030 [3]食品安全与营养协同创新中心,哈尔滨150028

出　　处：《中国食品学报》2015年第8期231-237,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金："十二五"国家科技支撑计划(2011BAD09B03)项目;食品安全与营养协同创新中心项目

摘　　要：文献显示:牛β-酪蛋白(CSN2)目前有12种遗传变种,提示人类中也可能存在这种现象。为探究中国人乳β-酪蛋白是否与NCBI数据库中该种蛋白的1级结构具有差异性,验证作者先前对中国人乳β-酪蛋白氨基酸序列的分析,采集正常中国女性乳腺组织,提取总RNA,并以其为模板,通过RT-PCR方法扩增人乳β-酪蛋白的编码区(CDS)基因,连接T载体测序,然后提交至Gen Bank(登录号:KF 751868)。与NCBI上人β-酪蛋白的核苷酸序列比较,所得人乳β-酪蛋白CDS序列同源性达到100%,说明该基因在不同人种间具有高度保守性。由其核苷酸序列及推导的氨基酸序列进行生物信息学分析,结果:理论等电点(p I)5.52,该蛋白质不存在跨膜结构,具有较强的疏水区域。连接重组质粒和PET-30a-GST载体后,转化大肠杆菌BL21(DE3)获得表达菌株,37℃诱导4 h,目标重组蛋白以包涵体形式表达,通过Ni2+-NTA柱纯化重组蛋白,经SDS-PAGE验证,成功构建原核表达载体PET-30a-GST-CSN2,并获得重组人乳β-酪蛋白。这为进一步研究中国人乳β-酪蛋白奠定良好的基础。It has been showed there are 12 genetic variants of β-casein in Bos taurus, and this kind of phe- nomenon might inherent in Homo sapiens. To confirm whether there was diversity between the primary structure of Chi- nese human β-casein and the data of NCBI database, and to verify the amino acid sequence of Chinese human β-casein that we reported previously. The total RNA extracted from normal breast tissue of a Chinese female was processed by RT-PCR to produce the human B-casein Coding Sequences region. The PCR product was cloned into vector T for se- quence analysis, this sequence was submitted to GeneBank database （accession number： KF 751868）. BLAST result of this sequence and the human B-casein CDS region published in NCBI database shows that the homology was 100%, such highly homology between different races suggests that this gene is conserved. The bioinformatic analyses and fore- casts of the nucleotide sequence and the deduced amino acid sequence were conducted： the theoretical pI was 5.52 and the protein contained no span membrane structures, and there existed high hydrophobicity region in the amino acid se- quence. The recombinant plasmid was cloned into the vector PET-30a-GST, induced by IPTG at 37 degrees Celsius for 4 h, the fusion protein expressed in Escherichia coli （E. coli） BL21 （DE3） mainly existing in the inclusion bodies. The recombinant human B-casein was purified using NI-NTA affinity chromatography, the result of SDS-PAGE indicated that the prokaryotic expression vector PET-3Oa-GST-CSN2 and the human recombinant CSN2 were gained. The whole work laid a solid foundation for the study of Chinese human B-casein.

关 键 词：Β-酪蛋白 基因 克隆 序列分析 

分 类 号：Q78[生物学—分子生物学] Q811.4