The Preparaion And Separation Of The Gecko Protein Composition

Objective To get out protein composition from gecko and separate the protein in different molecular mass.Methods Fresh gecko tissue was cut down, homogenated and freeze thawing for three times. Filtration, ultrafiltration, freeze drying methods were done after refrigerated centrifuge to get gecko protein crude. The gecko protein crude were dissolved, clear supernatant liquid was isolated and purified by G-50 Sephacryl gel chromatography. Different protein was collected according to separation curve and SDS-PAGE electrophoresis.Results Three protein peaks were got after gel chromatography collect the three protein peaks which were named A,B and C groups. The protein contents in the three groups was 44.8mg, 206.18mg, 126.38mg and the molecular mass was 20KD-50KD, 3KD-20KD and less than 3KD according to SDS-PAGE electrophoresis.Conclusions Three gecko protein compositions were got after gel chromatography, the molecular mass was 20KD-50KD, 3KD-20KD and less than 3KD,in which the content of B group was the highest. Objective To observe the suppression effect to hepatoma HepG-2 cells and leukemia K562 cells of the three gecko protein compositions in order to screen the best one.Methods MTS method was used to detected the suppression effect of the three gecko compositions in different density after 48h to hepatoma HepG-2 cells and leukemia K562 cells and to screen the group which with best suppression effect. Further to detect it's effect to the two cells after 72h. To observe the effect to cell morphological and apoptosis rate to the two cells after 48h by inverted phase contrast microscope and fluorescence microscope, calculate the rate of apoptosis.Results All of the three groups of gecko protein compositions could inhibit the growth of HepG-2 cells and K562 cells in different degree, the better effect with higher degree. 3KD- 20KD gecko protein group was more significant, it was statistically significant comparing with the control group. The two cells expressed cell morphological changes after 48h. The apoptosis rate were higher than the control group, it was statistically significant.Conclusions All of the three groups of gecko protein compositions could inhibit the growth of HepG-2 cells and K562 cells in different degree.3KD- 20KD gecko protein group was more significant, and the inhibiting effect exists dose-response relationship. The effect acted by inducing the apoptosis of HepG-2 cells and K562 cells. Objective To study the effect to c-myc,p53,bax and bcl-2 gene mRNA and protern after 48h of the 3KD-20KD gecko protein composition reacted to hepatoma HepG-2 cells and leukemia K562 cells.Methods The experimental group was HepG-2 cells and leukemia K562 cells effected by 3KD-20KD gecko protein composition and the control group was cells with no action. RT-PCR assay was used to detect the expression of bax, bcl-2, cmyc, p53 gene in the two groups. To the gene which was different, immunohistochemistry was used to detect the effect to protein expression.Results (1) RT-PCR assay showed that 48h later, the expression of bax gene increased in the two cells and bcl-2 gene decrease in K562 cells, it was statistically significant comparing with the control group. The expressions of cmyc and p53 gene showed no differences in exprimental group and it's not statistically significant.(2) Immunohistochemistry showed that 48h later, the expression of bax protein increased in the two cells and bcl-2 protein decrease in K562 cells, it was statistically significant comparing with the control group.Conclusions The 3KD-20KD gecko protein compositions induce the apoptosis of HepG-2 cells by enhancement of bax gene expression. And for K562 cells, it may involve enhancement of bax gene expression,attenuate of bcl-2 gene expression and the ratio change of bax/bcl-2.