Purification Of Tat-PTD-VP3 Fusion Proteins And The Research On Its Trans-Membrane Activity

Objective: To express the fusion proteins containing VP3 and Tat-PTD (protein transduction domain) proteins, and then purify and dialysis on this fusion proteins. To observe the trans-membrane activity of VP3 fusion proteins towards SGC7901 through the help of Tat-PTD. Meanwhile, to detect the position of the VP3 fusion proteins in this cells and its apoptosis effect on tumor cells.Methods: (1) Construction of the prokaryotic recombinant plasmid containing VP3 and Tat-PTD domain ,called pET32α(+)-Tat-PTD-VP3; (2) Transfected the recombinant plasmid into E.coli BL21 bacteria, expression the fusion protein Tat-PTD-VP3 through the effect of ITPG, tested its immunogenicity; (3) Inducing a large number of fusion proteins of Tat-PTD-VP3, disrupted the cells through ultrasound cell disruptor on the ice, then purified the fusion proteins through 50% Ni +-NTA column, and dialysised overnight in the PBS; (4)The purified protein and conventional cultured human gastric cancer cell line SGC7901 and umbilical vein endothelial cell co-cultured, observed the trans-membrane effet of the fusion proteins through fluorescence microscopy, and detected the apotosis of the cells through the TUNEL kit.Results: (1) We successfully constructed a recombinant vector containing Tat-PTD and VP3 gene, and enhanced green fluorescent protein EGFP also be cloned in this vector .The fragment was 1100bp after digested by EcoRⅠand XhoⅠ,consistanting with what we designed; (2) When we inducing the fusion proteins expression in the E.coli BL21 bacteria, we got a 42kD fusion proteins, about 20% of the bacterial total proteins; (3) Gray gel scanning showed that the purity of fusion protein is about 90% after purified by 50% Ni +-NTA ;(4) We have detected that the Tat-PTD-VP3 fusion proteins can be transferred into the cells in 30 min through fluorescence microscopy, and located in the nucleus of tumor sells; (5) TUNEL kit indicated that VP3 fusion proteins can significantly induce apoptosis of tumor cells.Conclusion: The purified Tat-PTD-VP3 fusion proteins have efficient trans-membrance activity , also can specific induce apoptosis of SGC7901 cells. The VP3 protein is a promising vaccine for the tumor treatment , and this experiment will be in favor of the research of anti-tumor treatment .