Study Of Gene Diversity And Biologic Activity Of Ricin

Ricin is a natural toxic protein isolated from the seeds of Ricinus communis.,which is one of the most powerful phytotoxin ever discovered. It consists of two polypeptide chains(A- and B-chain) linked through a single disulfide bond. Ricin A chain is an RNA-specific N-glycosidase that hydrolytically cleaved the N-glycosidic bond of the adenine residue at position 4324(A4324) within the 28S rRNA of eukaryotic cell and fatally disrupted protein synthesis in the end. Ricin B chain is an agglutinant,which can identify and bind terminal galactose residues on cell surface components. Ricin is one most toxin in the native poison up to now.It has been reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein synthesis inhibition. The toxicity of Ricin however has also been harnessed for use in medicine. In combination with monoclonal antibodies directed at cell surface receptors of tumourous cells, it fulfills the role of Erlich's magic bullet in the treatment of certain malignancies. Ricin is also used in the research of neurological degenerative disorders and in the treatment of intractable painful neuropathies by use of suicide transport in neurons.Ricinus communis is one of top ten oil crops. Our country is one of major cultivated region,and ricinus distributed in all parts of our country. Different variants of the ricin toxin and its amino acid sequence is not identical, In order to use ricin more rationally, we must understand their toxicities of each land races.Thus,we do this research about gene diversity and biologic activity of ricin. By the experiment, we found that:Cloned and sequenced RTA and RTB. Compared the sequences of RTA and RTB with the standard sequence X52908(Gene Bank).The results showed that :(1) RTA:36 nucleotide sites are different between No.03,04,05,09 and X52908, resulted in 18 amino acid sites mutation; 13 nucleotide sites are different between No.13 and X52908, resulted in 7 amino acid sites mutation; 2 nucleotide sites are different between No.16 and X52908, resulted in 1 amino acid site mutation; and no mutations occurred between No.01, 02, 06, 07, 10, 11, 12, 14, 17, 19 and X52908 both in nucleotide and amino acid sequences.(2) RTB: 26 nucleotide sites are different between No. 01, 02, 03, 04, 13, 20 and X52908, resulted in 15 amino acid sites mutation; 15 nucleotide sites are different between No.19 and X52908, resulted in 11 amino acid sites mutation; and no mutations occurred between No.05, 06, 07, 08, 09, 10, 11, 12, 14, 15, 16, 17, 18 and X52908 both in nucleotide and amino acid sequences. By analysis of sequences comparison result,No.02, 03, 05, 09, 10, 13, 16, 17, 18 were selected to continue the following experiment, as ricin extraction and purification, to compare their differences in toxicity.Ricin extraction and purification. Precipitate ricin in 70% ammonium sulfate saturation, purified ricin by Sepharose 4B agarose affinity chromatography column and Sephacryl S-200 gel filtration chromatography column. SDS-PAGE analysis,be only one band of 64kDa, Treated withβ-Mercaptoethanol and then SDS-PAGE,be two bands of 31kDa and 33kDa. Measured the concentration of 9 kinds of ricin,all about 10mg/ml.Toxicity experiments.(1) 9 kinds of ricin tomice administered by intraperitoneal injection,the results showed that :No.02, 03, 05, 09, 10, 13, 16, 17, 18 LD50 values are 19.81μg/kg, 44.11μg/kg, 39.83μg/kg, 41.82μg/kg, 20.88μg/kg, 48.22μg/kg, 19.52μg/kg, 21.13μg/kg, 19.21μg/kg respectively。(2)MTS assay 9 kinds of ricin toxicity on HeLa cells,the results showed that :the effect in the concentration of 0.01μg / ml on HeLa cells are basically in line; in the concentration of 0.1μg / ml the toxicity of No.05 was found weaker (* P <0.05);in the concentration of 1μg / ml, 10μg / ml and 100μg / ml No. 03, 05, 09, 13 was found significantly weaker than the standard sample and No.02, 10, 16, 17, 18(**P<0.01);and among the No. 02, 10, 16, 17, 18 and each of them compared with the standard sample are no differences.The study verified that domainⅠandⅡof RTA protein is the important regions to maintain its toxicity , the toxicity can be changed by transform the domainⅠandⅡof RTA. RTB mainly showed binding activity, the toxicity has no change though RTB gene mutations occurred in the study. In sum up, the study will provided an important theoretical basis for reasonable utilization of ricin.