Purification And Stabilization Of Reference Materials For Avian Leukemia And Pullorum Disease Detection

Avian leukemia and pullorum disease caused by avian leukemia virus and salmonella pullorum are two of most serious poultry epidemics that not only cause huge economic losses through the massive death of poultry,but also may infect human bodies and affect health.Therefore,effective prevention and control is vital to achieve complete elimination,but so far there is no effective vaccine.Diagnosis and cleaning out diseased chickens are the major strategy for prevention and control purposes.Rapid and accurate diagnosis of the desease is therefore extremanly important,but largely limited by lack of effective standard substances to ensure a reliable diagnosis results for poultry diseases.Developing diagnostic markers into reference materials can be used to calibrate existing detection techniques or detection kits,so that the results are traceable,comparable and accurate,and of great significance for the prevention and control of poultry diseases.Avian leukosis virus(ALV)antigen protein P27 and anti-Salmonella pullorum polyclonal immunoglobulin G(IgG)is generally used as a diagnostic marker for the coresponding desease.In this paper,we aimed at establishing purification and stabilization processes for these two diagnostic markers to be used as reference materials.The main results and findings are as follows:(1)A method for the purification of avian leukosis virus antigen protein P27 by two-step Ni affinity chromatography using his-tag was established.The his-taged P27 protein expreesed by recombinant E.coli was firstly purified by Ni affinity chromatography through bound-elution mode.After removing the his-tags on P27 by enzymatic digestion,the P27 was further purified by Ni affinity chromatography through a break-thrgough mode.After purification,the purity analyzed by SDS-PAGE was 99%and activity recovery measured by ELISA was 30%,respectively.Protein P27 degradation occurs in the preservation of the fermentation raw material and enzymatic digestion process was observed by tricine-PAGE and capillary electrophoresis with sodium dodecylsulfate(CE-SDS).Storing the raw material at-80℃ and adding protease inhibitor and imidazole in the enzyme digestion process was found effectively inhibit the degradation.The moledular weight of the final product determined by MALDI-TOF is consistent with the theoretical value,therefore both the traceability and purity satisfy the reference material preparation requirements.(2)A single step purification of polyclonal immunoglobulins G(IgG)against Salmonella pullorum from rabbit serum by anion exchange chromatography was developed.According to the difference of isoelectric point(pI)between IgG and the major impurities,the types of ion exchange media were systematically screened by comparison of their purification results.The results showed that pI of the IgG was 6.04-7.08 determined by capillary iso-electric focusing.In contrast,the purity of IgG was 99.3%and recovery rate was 67.5%after anion exchange chromatography with packing materials of Q-XL.After anion exchange chromatography with packing materials of Q-XL,a purity of 99%analyzed by SDS-PAGE and a recovery of 67.5%measured by high performance size-exclusion chromatography was achived under pH 5.5.In order to prevent the IgG from denaturation,different stabilizers were screened by differential scanning fluorimetry(DSF).200 g/L sorbitol was found to possess the best protection effect.The two thermal denaturation temperatures of IgG were increased by 5.52 and 8.84℃,respectively,and the stability at 70’C was significantly improved.The finally prepared polyclonal IgG has high purity,high recovery and high stability,and the preparation process is simple.It can be used for developing reference material to promote the prevention and control of pullorum disease.(3)O-antigen located at the outermost side of the Salmonella pullorum cell was extracted and its suitability as ligand of affinity adsorbent for IgG purification and antigen for indirect enzyme-linked immunosorbent assay(ELISA)of specific IgG anginst the bateria were explored.The O-antigen showed a specific affinity interaction with the anti-serum polyclonal IgG according to the isothermal titration calorimetry(ITC)analysis.By grafting the O-antigen onto the epoxy-activated giga-porous media,affinity adsorbent was successfully preapred,which showed a static adsorption amount of 0.46 mg-IgG/mL-media.When was coated on 96-well as antigen for serological testing,the O-antigen showed specificity in indirect ELISA.Under the optimal coating concentration of 40μg/mL,the lowest detection limit of IgG was 46.92 ng/mL,thus provided a powerful tool for accurate diagnosis of pullorum disease.