Clone And Expression Of A Lung Targeted Fusion Protein RGD-4C-His Tag-AGAP

The analgesic-anti-tumor peptide from Buthus martensii Karsch is the venom that has the activity of analgesia and anti-tumor.It is 192bp long and encodes 64 amino acid residues.As the damage to normal issues of anti-tumor drugs,targeted drugs have great dominance in therapy.So a fusion of lung cancer targeted AGAP was constructed.Since RGD-4C is a target and has an effect of repressing tumor metastasis according the literature,it is used as the target.Both Escherichia coli and Pichia pastoris have advantages and disadvantages in expressing hetero protein,so the fusion protein was expressed in both of them to compare: and find the better one.The sequence of the target was fused to 5’-terminal of AGAP through PCR.and then cloned into vector pNJU in which it is expressed in soluble form.Then the RGD-4C-His Tag-AGAP from supernatant of Escherichia Coli fragmentation was purified by means of metal chelated chromatogram.Pharmaceutical tests showed that the recombinant RGD-4C-His Tag-AGAP has an analgesic effect on mice in the animal model of twisting action of the mice induced by acetic acid.At the dosage of 3.78mg/kg,the peptide has the rate of inhibition 27.10%.A Pichia pastoris vector pPIC9K-RGD-4C-His Tag-AGAP was also constructed.It was lined and transformed into Pichia pastoris by electroporation,then linear DNA can generate stable transformants of Pichia pastoris via homologous recombination between the transforming DNA and regions of homology within the genome.The recombinant was induced by methanol,and the fusion protein RGD-4C-His Tag-AGAP was detected by western blot.