The Study On Ricin Biological Activity And Applied Research

Ricin is a toxic protein found in castor seeds,which are highly toxic, white powder, tasteless, can be dissolved in ethanol, ethyl ether, chloroform and benzene, acid and soluble salts in aqueous solution. It is composed of the All-toxic type of drug, lectin toxins and protein polymers. The types more crystalline were: B₁ ,T₃, G and D type, the small Ricin growth of China and Japan also contain E - ricin. D toxicity is the strongest. According to Ramesh, and other records isolated two kinds of different toxic protein RicinD and RicinE. While Cawley, and other isolated three kinds of different toxic protein Ricin 1, Ricin2 and Ricin3. These proteins isoelectric point ranged from 5.9 to 8.8. The protein toxin has a molecular weight of about 64 000, Molecular Formula is - [C₈H₈N₂O₂]_n-, which consists of two chains linked by a disulfide bond. RicinA chain (RTA) is a ribosome-inactivating protein,whose molecular weight is a bout 31000. RicinB chain is the binding subunit and can specifically recognize receptors on the cell membrane, which have a terminal galactose. RTB molecular weight is about 34000.The Ricin enters into the cell through receptor-mediated endocytosis. RTA binds to protein L3 of ribosomal 60S subunit, It has specific N-glycosidase activity, and can catalytically cleave a specific adenine at the site of 4234 out of the 28S rRNA.This cleavage of adenine can lead to deactivate the ribosomal function, thereby, inhibits the protein synthesis,and then cause the death of cells. For this reason, ricin has a good future for applying on anti-tumor Drug biological missile and biological pesticide.In this paper, have a study on methods optimization on how to extract Ricin from the Shelling castor bean. Selection of extraction methods are as follows: 5 mmol / L, pH6.5 phosphate buffer solution on Shelling castor bean defatted, and then use the phosphate buffer solution to extract the rough Ricins, with 60% ammonium sulfate to precipitate the crude Ricin proteins. Use the orthogonal experiments and the uniform design to optimize extraction Process conditions respectively, and combine the advantages of both methods to "uniformly dispersed, neatly comparable" properties to obtain the best conditions of the extraction process. At the same time used HI-experiment to verify the crude extracted ricin and found its still have biological activity.In the separation and purification step of Ricin, carryed out the obtained crude Ricin in 5 mmol / L, pH6.5 phosphate buffer solution dialysised 24 hours to desalting, concentrated its with polyethylene glycol 6000,then use Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration to isolate and purify the crude Ricin, then get the pure Ricin finnally. The isolated ricin samples in SDS-PAGE gel electrophoresis showed that the Ricin molecular weight is about 64,000, there are two subunits molecular weight of 31000 and 34000,the result is consistent with the reported references.Use the purified proteins to kill the Meloidogyne incognita and results showed that the ricin have strong contact toxicity in low concentration ,the Ricin lectin have strong stomach poisoning and contact toxicity activity. From the experiment we can see Ricin is the ideal raw materials to kill Meloidogyne incognita for biological pesticides.