Prokaryotic Expression Of Cucurmosin And Its Biological Activity Determination

Objective:The preparation process of Cucurmosin extracted from Pumpkin pulp is fussy and time-consuming.The cucurmosin has strong immunogenicity to the animals. To solve the above a series of problems, Preparation of recombinant pumpkin protein can be used by prokaryotic expression system which operation is simple and time-saving based on knowing the gene of the pumpkin protein. We may find more active fragment and reduce the immunogenicity of in the animals by selectively reducing amino acid codons for the mutants. It can provides a new method for antibody cross-linked by the cucurmosin gene site directed mutagenesis or increasing the amino acid for example cysteine.Method: Turn plasmid containing recombinant pumpkin protein gene into e. coli BL21(DE3) and screen positive expression colony to expand training. After using IPTG induced purpose gene expression, the results of expression were determined by SDS-PAGE. The recombinant protein can be purified by using nickel column affinity chromatography and further purified with ion exchange column.The pharmacological activities in vitro can be detected by SRB and MTT methods.we can detect the immunogenicity of Cucurmosin in mice By indirect ELISA.Result: Recombinant protein can be expressed by inclusion body form or soluble form. Because the renaturation efficiency of inclusions is very low, the soluble expression were obtained through the nickel column affinity chromatography method and acquired in 95% of the purified recombinant protein with ion exchange column. IC50 alue of recombinant pumpkin protein on pancreatic cancer cell PANC- 1, gastric cancer cells SGC- 7901,lymph cancer cell Raji,leukemia cell HL-60 were(6.0564±0.0014)μg/ml、(4.7697±0.0014)μg/ml、(9.9811±0.0018)μg/ml and(4.9640±0.0018)μg/ml.The antibody titer of recombinant Cucurmosin to mice is 1:640000.Conclusion:High purity reorganization of the pumpkin protein can be obtained by prokaryotic expression. Its pharmacological activity to pancreatic cancer cell PANC- 1 and Gastric cancer cells SGC-7901 in vitro is slightly better than the pharmacological activity of natural pumpkin protein. But its pharmacological activity to lymph cancer cell Raji and leukemia cell HL-60 is significantly weaker than the pharmacological activity of natural pumpkin protein. Its immunogenicity is slightly lower than natural pumpkin protein.