Study On Purification, Primary Structure, Immunoregulation And Anti-tumor Activity Of New Active Component Of Trichosanthin

Trichosanthin(TCS)is a typeâ… ribosome-inactivating protein(RIP) possessing N-glycosidase activity that was isolated from the root tuber of Trichosanthes kirilowii Maxim.From 1960s,a lot of researches have been progressed about TCS in basic theory and clinic application.TCS injection whose molecular weight was 26kDa has been found.Now its molecular structure,physical and chemical character and nucleotide sequence coding protein had been known.Otherwise,its gene can be expressed in E.coli and transgenic tobacco successfully.In recent years,TCS has also been found to posses various pharmacological properties including anti-early pregnancy and inducing mid-term abortion,immunomodulatory,anti-tumor and anti-HIV activities.TCS has been used to induce mid-term abortion and to treat ectopic pregnancies,hydatidiform mole and choricarcinoma in clinic.In recent years, TCS has been attempted to treat AIDS.At present,the methods to purify TCS including acetone precipitation,ion exchange chromatography,HPLC,Blue-Sepharose CL-6B chromatography and lot crystallization.But these methods were multiple,time-consuming and the purification procedure was easy to result in loss and influence yield.Our study consists of five sections:Sectionâ… :Purification of new active component of TrichosanthinIn this research,new active components of trichosanthin were purified from fresh trichosanthes kirilowii by ammonia sulfate precipitation,CM-52 ion exchange chromatography and G-75 gel filtration(molecular sieve chromatography).Thus,a convenient,quickly,high-performance purification method has been established.At the same time,the yields of these two new active trichosanthin components were compared.The conclusion is that the yields of these two new active components of trichosanthin from fresh wild type and artificial planting type trichosanthes kirilowii were different.Sectionâ…¡:Determination of molecular weight and N-terminal amino acid sequence of new active components of trichosanthinThe molecular weight was determined by MALDI-TOF MS analysis.The N-terminal amino acid sequence was determined by using the automated Edman degradation method.Furthermore,the molecular weights and N-terminal amino acid sequences of two new active components of trichosanthin from fresh trichosanthes kirilowii of wild type and artificial planting type respectively were compare with those of TCS injection and other high-homology proteins.The molecular weight of new active component of trichosanthin from fresh trichosanthes kirilowii of wild type was 33388.9, whose N-teiminal amino acid sequence was D-C-P-S-F-D-L-S-T-A-T-Q-D-S -Y-A-S-F-I-T-P-L-A.The molecular weight of new active component of trichosanthin from fresh trichosanthes kirilowii of artificial planting type was 32116,whose N-terminal amino acid sequence was D-C-P-S-F-D-L-S-T-A-T -Q-D-S-Y-A-S-F-I-T.The conclusion is that the molecular weights of these two new active components of trichosanthin from fresh wild type and artificial planting type trichosanthes kirilowii were different,but their N-terminal amino acid sequences were same.Otherwise,the molecular weight and N-terminal amino acid sequence of these two new active components of trichosanthin were different from trichosanthin injection(26000)and TAP29(29000)from American trichosanthes kirilowii.Sectionâ…¢:Effect on the immunoregulatory cells of new active component of trichosanthinTo investigate the effect of the new active component of trichosanthin on the immunoregulatory cells,we applied the flowcytometry to detect the percent of CD4+CD25+T cells of different mouse lymph organs after being immunized with the new active component of trichosanthin.The results showed that the new active component of trichosanthin could increase CD4+CD25+T cells of thymus and spleen.That means that the new active component of trichosanthin can suppress immune by increasing CD4+CD25+T cells.Sectionâ…£:Study on the anti-tumor activity of new active component of trichosanthinTo investigate the anti-tumor activity of the new active component of trichosanthin,we applied the flowcytometry to detect the effect of new active component of trichosanthin on the cell cycle and apoptosis of mouse melanoma cells(B-16),and MTT analysis to detect the growth suppression rate of mouse melanoma cells(B-16).The results showed that the new active component of trichosanthin could arrest B-16 cells in G₀/G₁ phase by increasing the cells in G₀/G₁ phase and decreasing the cells in S phase.At the same time,the new active component of trichosanthin induced the apoptosis of B-16 cells.The two effects will both be enhanced as the dose increasing.They are correlated highly (r=0.933,p＜0.01).Otherwise,the new active component of trichosanthin could suppress the growth of mouse melanoma cells(B-16).So the new active component of trichosanthin can suppress the growth of mouse melanoma cells (B-16).It also restrain the mitosis and induce the apoptosis of B-16 cells.The new active component of trichosanthin inhibits the B-16 cells by the above three ways.Sectionâ…¤:Study on the allergic reaction of guinea pigs to new active component of trichosanthin and trichosanthin injectionIn the allergic test of guinea pigs,we only found the first grade allergic reaction by using the new active component of trichosanthin to sensitize and attack guinea pigs,but we found the second grade allergic reaction by using the trichosanthin injection.The conclusion is that the allergic reaction of guinea pigs to the new active component of trichosanthin is negative.