Purification And Eukaryotic Expression Of Nerve Growth Factor From Naja Naja Atra

Objective To purify nerve growth factor of Naja naja atra and identify its purity and activity.To clone the gene encoding mature NGF from Naja naja atra and construct the eukaryotic expression vector pcDNA_3.1(-)-NGF and express it in mammalian cells NIH3T3.Methods Nerve growth factor of Naja naja atra was purified throught Sephadex G-75 column chromatography and CM Sepharose CL-6B cation exchange column chromatography. Liquor of positive reaction in every tube of eluting peak was collected after double immunodiffusion.Then identified the purity and activity by SDS-PAGE and PC12 cells.Total RNA was extracted from the venom gland using Trizol reagent. According to the cDNA sequence of Naja naja atra and the character of the expression vector,a couple of primers were designed and synthesized for RT-PCR amplification of the gene encoding mature NGF.The RT-PCR product and the eukaryotic expression vector pcDNA_3.1(-) were both digested by Kpn I and Xho I respectively,then reclaimed,ligated,transformed to construct recombinant expression vector pcDNA_3.1(-)-NGF.The recombinant plasmid was identified by the restricted enzymes and the sequence analysis. NIH3T3 cell was transfected with this vector using Lipofectamine^TM2000 transfection reagent.The expression of NGF in culture supernatant of positive clones was analyzed by SDS-PAGE and Western-Blot. Results Molecular weight of two samples, which were extracted from the cobra venom throught gel exclusion chromatography and cation exchange column chromatography, was 13KD and 23KD respectively.Two samples identified by PC12 cells both had activity of NGF.According to the restriction enzymes and DNA sequence analysis,the insert DNA was the gene which expressed the protein of cobra NGF.The NGF gene was expressed successfully in NIH3T3 cells.Conclusion NGF was successfully purified and had biological activity by identification.The gene encoding mature NGF was cloned from the venom gland successfully.The eukaryotic expression vector pcDNA_3.1(-)-NGF was constructed successfully.The NGF gene was expressed successfully in the transfected NIH3T3 cells by identification.