Cystatin M,Cathepsin B双基因共表达载体的构建及鉴定

Construction of vector of Cystatin M,Cathepsin B gene coexpression and sequence analysis

目的:构建半胱氨酸蛋白酶抑制剂M(Cystatin M,CST6),反义组织蛋白酶B(CB)单/双基因共表达载体.方法:从人胎盘组织中用TRIzol试剂提取总RNA,巢式PCR扩增人Cystatin M基因全长cDNA片段,RT-PCR扩增CB基因cDNA片段.将扩增的Cystatin M插入到真核表达载体pBudCE4.1的HindⅢ位点上,将CB插入到带有及不带Cystatin M基因真核表达载体pBudCE4.1的XhoI位点上.构建pBudCE4.1/CST6,pBudCE4.1/CB单基因表达载体及pBudCE4.1/CST6-CB双基因共表达载体.利用PCR、酶切分析和序列测定方法进一步验证所构建质粒的准确性.结果:人CST6,CB的RT-PCR扩增产物片段大小分别为447,257bp.对pBudCE4.1/CST6,pBudCE4.1/CB,pBudCE4.1/CST6-CB进行测序分析证实CST6,CB序列均正确,酶切鉴定及PCR结果显示,CST6,CB的大小分别为447,257bp且已克隆至pBudCE4.1中.结论:成功构建了pBudCE4.1/CST6,pBudCE4.1/CB,pBudCE4.1/CST6-CB表达载体,为深入探讨2种基因在肿瘤侵袭中的生物学作用机制奠定了基础.

AIM： To construct Cystatin M （CST6）, antisense Cathepsin B （CB） single/double gene coexpression vectors for the inhibition of tumor invasion. METHODS ： Total RNA was isolated from human placenta using TRIzol reagent. The full-length human cystatin M cDNA was amplified by nested polymerase chain reaction （nPCR） and Cathepsin B cDNA fragment was amplified by RT-PCR respectively. The cloned Cystatin M was ligated into the Hind Ⅲ cleavage site of the eukaryotic expression vector pBudCE4.1. Cathepsin B was ligated into the xho Ⅰ cleavage site of the pBudCE4. 1 vector containing the sequence for Cystatin M and that containing no Cystatin M sequence. Thus, pBudCE4.1/CST6, pBudCE4. 1/CB and pBudCE4. 1/CST6-CB were created. The recombinant vectors were further identified by DNA sequence analysis, PCR and restriction endonuclease digestion. RESULTS： Restriction endonuclease digestion analysis and PCR showed the expected 447 bp product for Cystatin M and 257 bp product for Cathepsin B. DNA sequencing showed 100% homology to the published sequence for cathepsin B and Cystatin M cDNA. This indicated that cathepsin B and Cystatin M cDNA had been cloned into expression vector pBudCE4.1. CONCLUSION： pBudCE4.1/CST6, pBudCFA. 1/CB and pBudCFA. 1/CST6-CB are successfully constructed, which will further help researches on the role of CST6 and CB in tumor invasion.