Purification Of Cystatin From Snake Venom And Expression Of Its Synthetic Gene In COS7 Cells

Objective1,To purify cystatin from Naja naja atra venom and to investigate its anti-tumor cell invasion in vitro.2,To investigate the expression of cystatin from snake venom in COS7 cells.Methods1,A inhibitor of cysteine proteinases was separated from naja naja venom by molecular sieve chromatography and affinity chromatography. The character of the inhibitor was analyzed by enzyme kinetics, HPLC and SDS-PAGE. The anti-tumor cell invasion potential of cystatin from snake venom in vitro was assayed in a Transwell cell culture chamber.2,According to amino acid sequence of cystatin from snake venom ,the full length of cystatin gene were artificially synthesized by slowly annealing PCR. The cystatin gene was amplified by PCR. The recombinant plasmids were constructed by inserting cystatin gene sequence into the plasmid pcDNA3.1/His C. COS7 cells were transfected with pcDNA3.1/His C-Cystatin by Lipofectamine and postive clones were identified by SDS-PAGE. The fusion protein was purified by ProBondTM purification system and identified by Western-blot.Results1,A low molecular weight cystatin was separated from naja naja venom by column chromatography. It can strongly inhibit the activity of papain by enzyme kinetics analysis and is identified a single component by HPLC analysis. 2,The cystatin can inhibit invasion of B16 melanoma cells in Transwell chamber.3,The recombinant expression plasmid pcDNA3.1/His C-Cystatin was constructed. The positive recombinant plasmid was screened and digested by two different endonucleases. The cystatin gene was successfully inserted into the eukaryotic expression vector pcDNA3.1/His C4,6×His-Cystatin fusion protein was expressed in the COS7 cells were transfected with pcDNA3.1/His C-Cystatin by SDS-PAGE analysis.The fusion protein was obtained by ProBondTM purification system. The molecular weight of the fusion protein was identifed about 15000 Dalton by Western-blot.Conclusions1,A low molecular weight cystatin was separated from naja naja venom ,and it can inhibit invasion of B16 melanoma cells in vitro.2,The recombinant cystatin protein in COS7 cells was expressed and purified.