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剂基因的启动子区。为此目的，我们已经检查了三种方法，已开发的已知结构基因的侧翼区的分离。用反向聚合酶链反应（PCR）和单特异性引物PCR方法，未能成功分离目的基因。用套式PCR方法可以分离到牛M1基因的一个侧翼区，用EcoR Ⅰ酶切牛肺DNA后，将DPIA片段的两端与EcoR Ⅰ盒连接。在EdoR 1盒引物C1和与牛M1基因第141 ~ 160位核苷酸部分互补的人工合成寡核苷酸的存在下，将扩增得到的DNA片段进行PCR。用EcoR Ⅰ盒引物C_2和与牛M_1基因第10 ~ 29位核苷酸互补的寡核苷酸进一步扩增产物DNA。从该反应中，我们可以分离到一个约500 bp的DNA片段。长度不进行DNA连接反应，不能获得该DNA片段。因此，500 bp的DNA片段应该夹在两个EcoRI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.