GGC54GAC MBL突变体的构建  

Construction of GGC54GAC mutant of mannan-binding lectin

作　　者：陈政良[1] 卢晓[1] 张丽芸[1] 韩强涛[1] 

机构地区：[1]第一军医大学免疫学教研室,广州510515

出　　处：《中国免疫学杂志》2001年第5期260-262,265,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金资助! (No .39970 2 86 )

摘　　要：目的 :在人甘露聚糖结合凝集素 (MBL)基因中引入GGC5 4GAC点突变。方法 :设计上下游引物和诱变引物 ,以汉族人野生型MBLcDNA为模板 ,采用megaprimer PCR技术进行定点突变 ,将PCR产物克隆至pGEM T载体 ,酶切和测序分析。结果 :以上游引物和诱变引物进行第一轮PCR ,获得一约 180bp的DNA片段 ,以此片段和下游引物行第二轮PCR扩增 ,得到一长约 780bp的产物 ;将其克隆至pGEM T载体 ,酶切发现第 5 4位密码中的BanI酶切位点已被破坏 ,与计算机酶切图谱分析结果一致 ;序列分析表明 ,除第 5 4位密码变为GAC外 ,余与野生型MBLcDNA完全相同。结论 :构建成功了GGC5 4GACMBL突变体 。Objective:To explore the mechanisms of mannan-binding lectin(MBL) deficiency,the GGC54GAC MBL mutant was constructed.Methods:The mutant was produced by the megaprimer PCR method for site-directed mutagenesis of wild-type MBL cDNA of Chinese,cloned into pGEM-T vector and identified by restriction analysis and sequencing.Results:With wild-type MBL cDNA as template and Deep Vent DNA polymerase,the first round of PCR reaction was performed using 5'-primer and the internal mismatch primer,which destroys a Ban I site in code 54 of wild-type MBL gene,and a product of about 180 bp was obtained.Then,the second round amplification was carried out using the fragment of 180 bp and 3-primer,producing a DNA fragment of about 780 bp.The last product of PCR was inserted into pGEM-T vector and a selected clone was identified by restriction analysis with Ban I and sequencing to show the expected mutation without any other change in the full length human MBL cDNA.Conclusion:The GGC54GAC MBL mutant was successfully constructed,which lays a foundation for investigating the mechanisms by which the GGC54GAC mutation of MBL gene causes immunodeficiency and the relation between structure and function of MBL molecule.

关 键 词：甘露聚糖结合凝集素 GGC54GAC突变体 定点突变 MBL 

分 类 号：R392.13[医药卫生—免疫学]