GGC54GAC MBL突变体的构建

目的：在人甘露聚糖结合凝集素（MBL）基因中引入GGC54GAC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，酶切了现第54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54位密码变为GAC外，余与野生型MBL cDNA完全相同。结论：构建成功了GGC54GAC MBL突变体，为深入探索MBL基因突变引起调理吞噬缺损的机制提供了分子病理模型。

Construction of GGC54GAC mutant of mannan-binding lectin

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.