Raji细胞基因组文库的构建与筛选  

Construction and screening of genomic library from Raji cells

作　　者：高建明[1] 李小玲[2] 李桂源[2] 

机构地区：[1]三峡大学医学院病理生理学教研室,湖北宜昌443002 [2]中南大学肿瘤研究所,长沙410078

出　　处：《中南大学学报（医学版）》2008年第3期185-191,共7页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金重点项目(30330560);国家自然科学基金项目(30470955)~~

摘　　要：目的：构建Raji细胞基因组文库，用EBV DNA探针对文库进行筛选。方法：Raji细胞基因组DNA经BamHI酶切消化后，低熔点琼脂糖回收9～23kb的酶切DNA片段，通过匹配黏性末端与磷酸化的Lambda DASH ⅡBam HI酶切载体臂连接，在体外经包装系统包装成活的重组噬菌体，测定原始文库与扩增文库的滴度，用同位素标记的EBVDNA探针对Raji细胞基因组文库进行筛选。结果：Raji细胞原始文库与扩增文库的滴度分别为1．8×10^5和2．8×10^8pfu／mL。文库经筛选获得4个阳性克隆，随机挑取1个阳性克隆稀释后铺平板作进一步杂交鉴定，发现所有噬茵斑均有杂交信号，证明该克隆确为阳性克隆。抽提该阳性克隆DNA，进行Bam HI酶切鉴定，证实插入片段的长度为8．5kb。经测序、BLAST序列比对，结果显示插入片段一侧为EBV BamHI W片段，另一侧与人类15号染色体RPl1-665A22克隆高度同源。结论：Raji细胞基因组文库的成功构建，为进一步克隆包含EBV整合位点的细胞基因组序列、探讨EBV整合参与肿瘤发生发展的分子机制奠定了基础。Objective To construct the genomic library of Raji cells and screen it by EBV DNA probe. Methods High molecular weight genomic DNA of Raji cells was digested by restriction enzyme BamHI. DNA fragments ranging from 9 to 23 kb were recovered by agarose gel electrophoresis, which were ligated with Lambda DASH II vector BamHI arms pre-treated with calf intestine alkaline phosphatase （CIAP）. Ligated DNA was packed in vitro using Gigapack m gold packaging extract. The library was plated on XLl-blue MRA （P2） host strain. Titering and screening of the Raji genomic library were performed. Results The primary titer of the Raji genomic library was 1.8×10^5 pfu/mL, while that of the amplified library was 2.8×l0^8 pfu/mL. Plaques （ 1 ×105 ） were screened with ^32p-labeled EBV DNA probe （EBV genome 5-3271 ）, 4 positive clones were obtained, and 1 of the 4 positive clones was picked out randomly for the second round of plaque screeninging. All the phage plaques were positive. DNA of the positive clone was extracted and was digested with BamHI. The length of the inserted fragment was 8.5 kb. Sequencing and BLAST analysis revealed that the inserted fragments consisted of the BamHI-W fragment at one end and clone RP11-665A22 on chromosome 15 at the other end. Conclusion The successfully established genomic library of Raji cells will provide a basis for cloning the sequences of the EBV junction sites and inter- preting the mechanism of oncogenesis of EBV integration.

关 键 词：RAJI细胞 基因组文库 构建 筛选 

分 类 号：Q78[生物学—分子生物学]