Construction of vector of Cystatin M,Cathepsin B gene coexpression and sequence analysis
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% homol-ogy 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 pBudCF4 1. CONCLUSION: PBudCE4.1/CST6,PBudCE4. 1/CB and PBudCE4. 1/CST6-CB are successfully constructed,which will further help researches on the role of CST6 and CB in tumor invasion.
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NSTL
万方数据
维普
