Expression Of Recombinant Curcin And Its Antitumour Activity In Vitro

Objective:To amplify curcin gene from the genomic DNA of Jatropha curcas and express it by E.coli, and to establish the technology of purification and renaturation of recombinant curcin. To study the effects of recombinant curcin on proliferation and apoptosis of tumor cells.Methods:1. Amplification of curcin gene:The total DNA was extracted from the leaves Jatropha curcas. Using the Jatropha curcas total DNA as template, curcin gen was amplified by PCR.2. Cloning and Expression of curcin gene:The curcin coding fregment was inserted into plasmids pET22b and recombinant plasmid pET22b-curcin was constructed. Recombinant plasmids pET22b-curcin was transformed into E.coli OB. Using IPTG to induce expression of the recombinant protein.3. Optimization of expression conditions:The effects of various concentration of revulsants, inducing temperature and revulsant adding time on the expression of recombinant protein were studied.4. Purification of recombinant protein:After shaking flask fermentation, recombinant bacteria were collected by centrifugation, and then disrupted by ultrasonic. The inclusion bodies were collected by centrifugation, and then resuspended with 2M urea, ultrasonic washing. The suspension was dissolved by 8M urea. The expression product was purified by Ni2+ affinity chromatography (equilibrium liquid:8M urea; washing liquid:2M urea,30 mM imidazole and 50 mM imidazole; eluent:300 mM imidazole). And the Sphacryl S-100 gel-chromatography was used for the further purification.5. The effects of recombinant curcin on proliferation of tumor cell:The MTT assay was used to detect the proliferation inhibition of recombinant protein on some tumor cells, i.e ke A549, HepG2, MDA-MB-231, CNE-2Z, SPC-A-1, Lewis, hela, smmc-7721, MCF7 and SKBR23 cells. Apoptosis assay was used for the detection of tumor cell apoptosis.6. The effects of recombinant curcin on apoptosis of tumor cell:After treated with recombinant curcin, A549 cells were stained with acridine orange, observed under the fluorescence microscope. Results:1. The total DNA was extracted from the leaves of Jatropha curcas successfully.2. Using the total DNA as template for PCR, curcin gene was amplifid successfully. Alignment result showed that the homology between our sequence and the sequence of GeneBank database was 98.6%.3. Recombinant expression plasmid PET22b-curcin was constructed successfully. Induced with 30℃,6h, the recombinant curcin that molecular weight is about 29kDa was expressed in recombinant strain transformed with PET22b-curcin. The expression level of recombinant curcin was about 42% of the total cellular protein.4. The result of soluble assay indicated the recombinant curcin was expressed in the form of inclusion body.5. After purified by Ni2+ affinity chromatography and Sphacryl S-100 gel-chromatography, the purity of the recombinant protein was 95.6%, the yield rate was 27.5mg/L and the total recovery was 65.6%.6. MTT assay shows that the recombinant curcin can significantly inhibited proliferation of A549, HepG2, Lewis, MDA-MB-231, CNE-2Z, SPC-A-1, MCF7 and smmc-7721 cells. But the proliferation inhibited effects on hela and SKBR23 cells were not significant. Apoptosis assay shows that recombinant curcin promoted apoptosis of A549.Conclusion:1. Successfully isolated and cloned curcin gene from genomic DNA of the local Jatropha curcas, and expressed in the prokaryotic expression system.2. Optimized the conditions of high level expression recombinant protein in E.coli, established the inclusion body purification, renaturation process, purified recombinant protein yield rate was 27.5mg/L.3. Experimental results in vitro showed that curcin recombinant protein's inhibition of tumor cells were different; Apoptosis experiments confirmed that curcin recombinant protein had the ability of promoting tumor cells apoptosis.4. Curcin recombinant protein has potential applications on some types of cancer treatment.