The Study Of Bioactivities Of Grimin Gene From Lampetra Japonica

Grimin roots in the anti-tumor gene recombination protein GRIM-19 of Lampetra japonica buccal gland, where GRIM-19 is a kind of the cell death activation factor which was recently discovered. Because the tumor cell transmission can be blocked by the GRIM-19's specific suppressor to the rule of the STAT3 which stimulate the tumor cell apoptosis or inverts malignant phenotype, and the Grimin of Lampetra japonica has the same functional domain and the higher sequence homology with GRIM-19. So it hopefully becomes the anti-tumor genetic engineering new medicine with powerful effects, little poison and little negative factor.In view of it, we has withdrawn successfully the gene with the same function as GRIM-19 from Lampetra japonica. It has been conducted the preliminary research and the corresponding confirmation,named as Grimin.Our team have finished the constructing work of the gene library about Lampetra japonica buccal gland's cDNA with its 2.1×10⁶pfu·ml^-1 capacity. We have withdrawn the total RNA from the Lampetra japonica in its migration to Heilongjiang basin, using our independently design premier to carry on the RT-PCR expanding, and have obtained the new function gene of 429bp long Grimin. The premier design is basised on the GRIM-19 homology protein sequence of disintegrins published on NCBI, which deduces the DNA sequences and designs two oligonucleotide primers. the result shows, this function gene and the African toad GRIM19 homology is 66%, and the globefish GRIM19 homology is 68%.The paper follows the above works , and the histidine labeled pET23b-Grimin has been transformed into expresses in fungus E.coliRosetta to carry on the IPTG primer expression; after the affinity column analysis of histidine to purify the expressed reorganization protein. The active protein is obtained after denaturation and renaturation of its product inclusionbody, and named Grimin.obtained the biological activity Grimin protein, we have further carried on the partial biological activity examination to it. Through Raising personal vena umbilicus endothelial cell ECV304 and personal leukemia cell HL60, collecting cell , using the MTT color method, the double benziminazole (Hoechst 33,258, Ho) dyes and flows experiment analysis, the Grimin protein has been proved its biology activeness which can suppress tumor cell grows and promote it apoptosis; Moreover we has carried on the little experiment of the lipide body peridium about Grimin, which enables the Grimin peridium protein penetration cell membrane to function in the cell. The experiment make the separately comparison with the cell added protein and the cell not added the protein only plus lipide cell; the results show the cells with lipide protein wraped appear the obvious perish and lipide body does not enduce any stimulation to cell. Thus it proves that the lipide body peridium may be taken as the anti-tumor medicine dosage to apply.In the paper we successfully obtain the new functional gene and protein from Lampetra japonica through the genetic engineering method to study the GRIM-19 induce tumor cell apoptosis, and make some preliminary research to the relative biology activity about Grimin. These experimental results can prompt Grimin the latent application value to be taken as one kind of catalyst inducing tumor cell to death.