脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA

Cloning, Sequencing and Site-directed Mutation of Lipoprotein Lipase Gene

The total RNA was isolated from the human fat tissues.According to the reported cDNA sequence of the lipoprotein lipase (LPL),primers were designed and synthesized.By means of RT\|PCR, one PCR fragment (1 6 kb) was obtained.The PCR products were cloned into pGEM\|T vector.Three clones were sequenced.Analysis of the nucleotide sequences showed that they were the same as that of the LPL cDNA.The polymorphisms of LPL gene were detected by denaturing high performance liquid chromatography(DHPLC) in coronary heart disease (CHD) patients, and a novel point mutation G830A within the fifth exon was found,which changed the 192 codon CGA into CAA and resulted in the substitution of glutamine for arginine.Site\|directed mutation based on PCR was used to produce the varied LPL cDNA,and another two primers surrounding the varied nucleotide were synthesized and the altered nucleotide was included in one of the primers.Two PCR fragments (843 bp and 811 bp)were ligated and their product was cloned into pGEM\|T vector,whose sequence was confirmed by DNA sequencing.