Molecular cloning of collagenase inhibitor gene from bovine genomic DNA

本研究的目的是分离牛胶原酶抑制基因的启动子区域。为此，我们研究了三种分离已知结构qenes侧翼区域的方法。用反向聚合酶链式反应和单特异性引物聚合酶链式反应的方法，都没有成功地分离出目的基因。但用套式聚合酶链式反应方法可以分离出牛m1基因的侧翼区，用EcoRl‘酶切牛肺DNA后，将DPIA片段两端与EcoRl盒连接。所得DNA片段在EdoR1盒引物盒的存在下进行聚合酶链式反应，合成的寡核苷酸与牛M1基因的一部分互补，其大小为141~160个核苷酸。用EcoRl盒引物C_2和另一个与牛m1基因第10~29位核苷酸互补的人工合成的寡核苷酸进一步扩增产物DNA。从这个反应中，我们可以分离出一个约500bp的DNA片段。长度。如果没有DNA连接反应，就不能获得该DNA片段。因此，500bpDNA片段应夹在两个EcoRl盒和牛m1基因的上游区域之间。

The purpose of this study was to isolate promoter regions of bovine collagenase inhibitor genes. For this purpose, we have examined three methods which have been developed for the isolation of flanking regions of known structure qenes. With inverse polymerase chain reaction(PCR)and single-specific primer PCR methods, it was unsuccessful to isolate a target gene. However, we could isolate a flanking region of bovine Ml gene by usinq nested PCR method.After digestion of bovine lung -DNA with EcoRl', the DPIA fragments were ligated with EcoRl cassette at both ends. The resultment DNA fragments received PCR in the presence of EdoRl cassette primer Cl and a synthetic oligonucleotide complementary with a part of bovine Ml gene which spans from 141th to 160th nucleotides. The product DNA was further amplified by using EcoRl cassette primer C_2 and another synthetic oligonucleotide complementary with a part of bovine Ml gene which spans from 10th to 29th nucleotides. From this reaction, we could isolate a DNA fragment with about 500 bp in. length. This DNA fragment could not be obtained without DNA ligation reaction. Thus, the 500 bp DNA fragment should be sandwiched between two EcoRl cassettes and a upstream region of bovine Ml gene.