一种BAC载体的构建及其在CYP2A6单倍型分析中的应用  

Construction of a BAC Vector and Its Application for Haplotype Analysis of CYP2A6

作　　者：赵芳玲 綦钰莹 蔡剑平 戴大鹏 ZHAO Fangling;QI Yuying;CAI Jianping(Peking University Fifth School of Clinical Medicine,Beijing 100730,China)

机构地区：[1]北京大学第五临床医学院,100730 [2]国家老年医学中心、国家卫生健康委北京老年医学研究所、国家卫生健康委北京老年医学重点实验室、中国医学科学院老年医学研究院,100730

出　　处：《医学研究杂志》2024年第2期24-28,111,共6页Journal of Medical Research

基　　金：国家重点研发计划项目(2020YFC2008301);国家自然科学基金资助项目(面上项目)(81971323)。

摘　　要：目的构建大片段基因克隆用高拷贝BAC质粒载体以用于CYP2A6基因的单倍型分析。方法利用退火程序生成包含多克隆位点的寡核苷酸,分别连接入HindⅢ/BamHⅠ双酶切后的pGEM-3Z载体及pBeloBAC11载体,随后使用HindⅢ单酶切中间载体并连接,利用蓝白斑筛选获得头尾串联的高拷贝BAC载体pBAC-BJH。利用STI PCR的方法扩增CYP2A6基因全长,经切胶纯化后利用BstBⅠ与MluⅠ双酶切,并与同样双酶切的pBAC-BJH载体连接,转化大肠杆菌以获得包含新突变355A>T的全长Bac克隆用于单倍型测序分析。结果成功构建了增加7个多克隆酶切位点的高拷贝BAC载体pBAC-BJH。该载体具有拷贝数高、可选酶切位点多等优势。利用该载体对CYP2A6的基因分型结果显示,新突变体携带22C>T、51A>G、355A>T,3个SNP单倍型为CGT。结论成功构建了方便用于大片段基因克隆的载体pBAC-BJH,该载体可用于p450基因的单倍型分析及其他大片段基因克隆分析。Objective To construct one high-copy BAC vector with cloning capacity for large DNA fragment insertion and use it for the haplotype analysis of CYP2A6 gene.Methods Oligos including multiple cloning sites were annealed and ligated into the HindⅢ/BamHⅠsite of pGEM-3Z and pBeloBAC11,separately.Then,the intermediate vectors were digested by HindⅢand ligated together,so as to get the head-to-tail oriented high-copy BAC vector pBAC-BJH after the blue-white spotting test.STI PCR method was used for the amplification of whole CYP2A6 gene,and purified amplicon was double digested by BstBⅠ/MluⅠand ligated into the same site of newly constructed vector pBAC-BJH.Several clones were then picked up and sequenced for the haplotype analysis of carriers with newly discovered CYP2A6 variant 355A>T.Results One high-copy BAC vector pBAC-BJH with 7 newly added multiple cloning sites was successfully constructed.High copy number and multiple cloning sites were the advantages of this plasmid.With this vector,haplotype analysis result for 22C>T,51A>G,355A>T in carrier with newly detected CYP2A6mutation was CGT.Conclusion One vector pBAC-BJH convenient for cloning large DNA fragment was successfully developed,and it can be used for the haplotype analysis of cytochrome p450 gene and cloning or sequencing of other genes with large genomic DNA inserts.

关 键 词：高拷贝 BAC载体 CYP2A6 单倍型分析 

分 类 号：R393[医药卫生—基础医学]