| Ricin is a glycoprotein extracted from the castor oil plant Ricinus communis. Ricin content of castor beans probably is in the range of 1% to 3%. Ricin has a molecule weight of 64 kDa and consists of two glycoprotein chains active A and B that are linked by disulfide bond. RTA is effects chain and RTB is combined chain. RTA molecule weight is 64 kDa, composed of 267 amino acids. Active conservative sites Glu177 and Arg180 which are located in the catalytic center is essential to the RTA's activity. RTB consists of 256 amino acids and molecular weight is about 34kDa, its two oligosaccharide chains (GlcNAc)2(Man)8 and (GlcNAc)2(Man)7 are connected to the 95 and 135 Asn residues, if removing these two oligosaccharide chains, can lead to loss of RTB lectin activity.Ricin is biological terrorism-related protein toxin and currently no vaccine or specific countermeasures is available for ricin poisoning. Because it is lethal and available worldwide, it is considered a likely biological agent for bioterrorism. Ricin also has significant anti-tumor effect, the immunoconjugate composed by ricin and anti-tumor specific McAb has a good prospect. Therefore, to study ricin not only have important military significance, but also have great clinical value.In this study, we searched the extraction and purification ricin, preparation of monoclonal antibodies against ricin toxin, we established GICA, HPLC, SPR detection method and constructed recombinant ScFv anti-ricin prokaryotic expression vector. The main results are as follows:⑴In the separation and purification of ricin, ricin and ricin agglutinin were adsorbed by Sepharose 4B column, then used Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration to isolate ricin and ricin agglutinin. RTA was about 32kDa, RTB was about 34kDa. RT was administered by Kunming mouse tail vein injection. Then mice LD50 was measured by modified Kou's method. After 48h, the LD50 was 1.20±0.26μg/kg, 72h of the LD50 was 0.71±0.15μg/kg. After 12h, the cytotoxicities of RT on Hela and mouse macrophage cell line RAW246.7 cells were measured by MTT assay. The concentrations causing 50% cell death (IC50) of RT in Hela and RAW246.7 cells were 9.39ng/mL and 35.27ng/mL respectively. The IC50 of these two kinds of cells was between 0.05-0.01ng/mL after 24h. RT's pI (isoelectric point) measured by thin-layer isoelectric focusing was 7.10.⑵Six strains of anti-RT McAb were screened, then were named as 1H4, 4D8,4D12,1C,2F,3A. Six monoclonal antibody titers in ascitic fluid were: 3.7×105, 1.19×107, 4.5×106, 3.2×106, 5.7×106, 2.6×106; Identificated by indirect ELISA: 1H4, 4D8, 4D12, 1C, 2F, 3A were both at the IgG1 subclass; Epitope analysis showed that: 1H4, 4D8, 4D12 monoclonal antibody antigenic sites were in ricin A chain. 1C, 3A monoclonal antibody antigenic sites were in the ricin B chain, 2F monoclonal antibody in the ricin A, B chain had its antigenic sites; Affinity constants and dissociation constants that 1C, 1H4, 2F, 3A, 4D8, 4D12 and other McAb coupled with ricin were: 1C of KA =1.86×1010M-1, KD=5.38×10-11M; 1H4 of KA=2.29×108M-1, KD=4.36×10-9M; 2F of KA=4.1×108M-1, KD=2.44×10-9M; 3A of KA=1.93×1010M-1, KD=5.18×10-11M; 4D8 of KA=3.40×1013M-1, KD=2.94×10-14M; 4D12 of KA=1.81×1011M-1, KD=5.53×10-12M.⑶Two different monoclonal antibody with anti-RT different antigenic sites was used as the gold standard, an antibody as the gold standard, the use of colloidal gold immunochromatography assay for RT was explored. 40nm colloidal gold labeled monoclonal antibody was prepared; The process conditions of strips was optimized, The test strip was assembled; standard test paper was used to detect toxins and simulated samples, the results show that the strip is the minimum detection limit was about 10μg/mL, detection time was 5-10min; specificity and stability of the test strip were studied.⑷Using the Showa Corporation PROTEIN KW-802.5-gel column (8×300mm), with 0.3M NaCl 0.05M phosphate buffer (pH=7.3) as mobile phase, detection wavelength of 280nm, established a RT HPLC detection method. In the range of 0.093-5.97μg ,the linear regression equation was Y=0.0116X+34.63, R2=1, the standard rate was 102.39%±5.06%, coefficient of variation was 4.94%; Ricin recoveries concentrated in water and unconcentrated were 101.10%±1.16% and 93.04%±1.23%, coefficients of variation were 1.15% and 1.32%; Ricin was added in purified water and sausage samples, the average recoveries were 94.32±0.74% and 80.99±1.66%, coefficient of variation were 0.78% and 2.05%; the assay limit of RT detection was 23.33ng.⑸A method of SPR was established to detect ricin, within 0-2000ng/mL the linear correlation coefficient R2=0.9887, the lowest detectable concentration was 0.05ng/mL. SPR technique was developed to identify abrin. In the range of 250-2000ng/mL, the linear correlation coefficient R2=0.9886, the lowest detectable concentration was 0.5ng/mL.A mixture of the two toxins at the same time was detected as with the individual results.⑹using genetically engineered antibody technology, ScFv Prokaryotic expression vector was constructed and expressed, then we investigated its biological activity: antibody light and heavy chain variable-region genes was obtained by PCR amplification from the 4D12 hybridoma cell line which secrets anti-ricin monoclonal antibody and the nucleotide sequence was determined. VL and VH were spliced into ScFv gene by (Gly4Ser)3Linker gene. Sequencing results showed that the gene is 750bp and VL-Linker-VH was spliced correctly; The expression vector pMAL-p2X was used for soluble expression of ScFv fragments. Anti-RT ScFv was obtained by Amylose affinity chromatography purification; ScFv activity was measured by ELISA and SPR, the results showed that single-chain antibodies against ricin stability combined with ricin. RT ScFv binding kinetics detected by SPR biosensor: KA=1.69×108 M-1, KD=5.93×10-9M.
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