七种常见MBL基因单倍型标准质粒的构建

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (11): 1379-1383.

七种常见MBL基因单倍型标准质粒的构建

余新沛, 陈月, 张丽芸, 卢晓, 陈政良

南方医科大学免疫学教研室, 广东, 广州, 510515

出版日期:2005-11-20 发布日期:2005-11-20
基金资助:
收稿日期:2005-7-13。
基金项目:广东省自然科学基金研究团队项目(015003);广东省科技攻关项目(C30201);广州市重点科技攻关项目(2003Z2-E4031);广东省“十五”重大科技专项(2004A30801005)
作者简介:余新沛(1978-),男,硕士研究生,主要从事天然免疫研究
通讯作者:陈政良,Tel:020-61640114-89280

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

YU Xin-pei, CHEN Yue, ZHANG Li-yun, LU Xiao, CHEN Zheng-liang

南方医科大学免疫学教研室, 广东, 广州, 510515

Online:2005-11-20 Published:2005-11-20

摘要/Abstract

摘要： 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。

Abstract: 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 of exon 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 of MBL gene, the standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA and LYPB of MBL 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 of MBL gene with such genotyping methods us SSP-PCR and real-time PCR.

中图分类号:

Q319
R392.2

余新沛, 陈月, 张丽芸, 卢晓, 陈政良. 七种常见MBL基因单倍型标准质粒的构建[J]. 南方医科大学学报, 2005, 25(11): 1379-1383.

YU Xin-pei, CHEN Yue, ZHANG Li-yun, LU Xiao, CHEN Zheng-liang. Construction of standard recombinant plasmids for 7 common mannan-binding lectin gene haplotypes[J]. Journal of Southern Medical University, 2005, 25(11): 1379-1383.

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