In Vitro Expression Of RIPs Of Bougainvillea Glabra In Pichia Pastoris And Antitumor Activity Analysis Of The Recombinant Protein

Plant sources of ribosome-inactivating proteins(RIPs)is a kind of RNA N-glycosidase widely exsited in advanced plants,it can take off the adenine of ribosomal RNA on the large subunit of ribosome,thus destroying the structure of ribosome and inhibit protein biosynthesis.In application,the immunotoxin constructed with ribosome-inactiving protein has a promising prospect in the treatment of cancer.Bouganin is a type I RIPs first isolated from the Bougainvillea.glabra Wild,which is the lowest toxic RIPs found at present.The laboratory used homologous sequence colning method cloned DNA from the Bougainvillea glabra choisy in xiamen city and has expressed using Escherichia coli prokaryotic expression system.However the recombined protein expressed by E.coli mostly exist in the form of inclusion body,it is difficult to purification,and the generated Bouganin-specific immune antibodies is not highly efficent.With amplfied Bouganin gene,pichia pastoris GS115,the vector pPIC9K plasmid,we constructed the GS115-pPIC9K-Bouganing expression system.Then we optimized the expression condition.Finally the recombined eukaryotic Bouganin protein(Bouganin)was purified by immobilized metal ion afinity chromatography and tested the antitumor activity by MTT assay.The research showed that:(1)the subphenotype of the transformed GS115 is muts,indicating the promtor AOX2 acted;(2)The best expression time is 48h and the applicable temperature is 22?;(3)Bouganin secreted in the medium,easy for purification,but the expression level wasn't high as expected,which is 0.225mg/L;(4)The results of the anti-tumor test showed that the antitumor activity of eukaryotic Bouganin protein was better than that of the prokaryotic Bouganin protein.When the concentration of Bouganin was 100 ug/mL,the inhibition rate of the eukaryotic Bouganin protein in Hela,HEP3B and A549 cells was 27.75%,18.05%and 20.68%respectively,while the inhibitory rate of the prokaryotic Bouganin protein was 20.27%,6.84%and 13.79%respectively.After univariate analysis of variance,there was no significant differencebetween the inhibition efficiency of eukaryotic and the prokaryotic Bouganin protein on the tumor cells.