七种常见MBL基因单倍型标准质粒的构建

Construction of standard recombinant plasmids for 7 common mannan-binding lectin gene haplotypes

目的构建7种常见的MBL基因单倍型标准质粒。方法以已确认MBL基因单倍型及基因型的DNA样本为模板,利用SSP-PCR技术扩增出包含MBL基因启动子区及第一外显子的单倍型片段并将其克隆入T载体;用定点突变技术分别将其第一外显子52和57位密码相应的碱基突变;采用PCR和测序进行鉴定。结果以单倍型及基因型已确认的DNA样本为模板,采用SSP-PCR和分子克隆技术获得单倍型HYPA、LXPA、LYQA、LYPA和LYPB重组质粒;以单倍型HYPA及LYQA重组质粒为模板,采用定点突变技术获得单倍型HYPD及LYQC标准质粒。结论构建成功常见的7种MBL基因单倍型标准质粒,为采用SSP-PCR、Real-timePCR等技术分析MBL基因相应的SNP及其单倍型与基因型提供了标准对照。

Objective To construct the standard recombinant plasmids for 7 common haplotypes of mannan-binding lectin （MBL） gene. Methods The DNA samples with known haplotypes and genotypes of MBL gene were used as the templates for amplifying the fragments of MBL gene haplotypes including the promoter region and exon 1 with sequence-specific primer-polymerase chain reaction （SSP-PCR） method. The amplified fragments were cloned into T vector and the bases located at codon 52 and codon 57 ofexon 1 in MBL gene were mutated respectively by site-directed mutagenesis. All the 7 recombinant plasmids were identified by PCR and direct sequence analysis. Results From the DNA samples with known haplotypes and genotypes ofMBL gene, the standard plasmids ofhaplotypes HYPA, LXPA, LYQA, LYPA and LYPB ofMBL gene were constructed by SSP-PCR and molecular cloning technique. From the recombinant plasmids of HYPA and LYQA, the standard plasmids of haplotypes HYPD and LYQC of MBL gene were constructed by site-directed mutagenesis, respectively. Conclusion The constructed standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA, LYPB, HYPD and LYQC of MBL gene provide standard controls for detecting the SNPs, haplotypes and genotypes ofMBL gene with such genotyping methods us SSP-PCR and real-time PCR.