Cloning,Expression Of BTI Gene And Primary Research Of Biological Function

Apoptosis is a cell initiative death course, that caused by cell's internal and external environment changes, death signal touching off and the regulation of gene. This course plays an important role for dispelling the aging cell in the organism or having potential cell that unusually grew, and keeping the organism being in homeostasis. The disorder of internal apoptosis regulation and control mechanism will cause human body many kinds of illnesses. The formation of tumors and the senile dementia belong to this kind of typical illness. A lot of researchers at present focus on study on protease and protease inhibitor in the cell and they think these moleculars might play a key role in apoptosis.This paper regarded Shouyang buckwheat as studied materials. TotalRNA which was isolated from the leaves of plants was reversely transcribedinto cDNA. Buckwheat trypsin inhibitor gene was gotten through numerousPCR with designed primers which had enzyme cut site according to thesequence of expressive vector. Then PCR product was connected topGEM-Teasy vector after amplifying. After verification, the purpose genewas cut from the plasmid of pGEM-T-BTI by BamH I and Hind Ⅲ and wasconnected with the expressive vector pQE31 that was cut by the sameenzymes. The constructed plasmid was transformed into the host cell ofE.coli M15[pREP4]. Through optimizing the expression condition, it wasconsidered appropriate to expressive the target protein with OD60o=0.5-0.6, 0.5mmol/L IPTG, 37°C, and express for 4 hours. The expressed product could be purified through HiTrap Chelating HP column, and be eluted by the elution buffer contianing 0.5mol/L imidazole. Target protein was identified by Tris-Tricine SDS-PAGE and Western blot, and the recovery rate and yield were estimated by means of the analysis of BTI activity.The result of study showed that we constructed successfully pQE31-BTI expression vector, and purified the expressed product. After identification, the target protein had been dried in frozen state. The inhibitory activity of recombinant BTI was about 77.16 Ui/mg. The recovery rate could be up to 88. 56%.Because the protease inhibitor can suppress the growth of the tumour cell, it is regard as a kind of potential antineoplastic factor. The recombinant protein could induce apoptosis of tumour cell. At first its biological activation of suppressing tumour cell to grow was determined with MTT assay, and then its inhibitory rate growing to tumour cell was determined with flow cytometry.The recombinant BTI could suppress growth of IM9 cell through MTT assay. Result showed that recombinant BTI had strong restrain function of growth to IM9 cell and IM9/ Bcl-2. Up to 27.56% IM9 cell was restrained, and when IM9 cell expresses Bcl-2 at the same time, 14.46% IM9/ Bcl-2 cells were strongly restrained. It was shown that the recombinant BTI could...