| The Nile tilapia (Oreochromis niloticus) is an economically important species worldwide. In recent years, diseases that resulted in huge financial loss in tilapia aquculture are increasing due to the expansion of the cultivation scale. Pool Sterilizing and treating with antibiotic can remit disease to some extent, but can not resolve the problem fundamentally, even cause environmental pollution owing to the antibiotic residual in the fish. Fish possess an efficient and developed immune system and Immunoglobulin (Ig) play an important role in humoral immune response. Studying serum Ig of tilapia will contribute to understanding the process of immune response, which can provide theoretical basis for prevention of diseases.Monoclonal antibodies (mAbs) have been applied to many fields including fish immunology due to their strong specificity for binding antigen. mAbs against serum Ig of tilapia were powerful tool for studying of tilapia immune system, purification of tilapia serum Ig is the essential step for preparing of mAb. In this study, serum Ig of tilapia was purified and mAbs against serum Ig of tilapia were produced. Characteristics of mAbs obtained in this study were investigated, and then the mAbs were applied to explore the dynamics characteristic of specific antibody of tilapia against Aeromonas hydrophila. The main results are as follows:1. Serum Ig from tilapia was purified by Sepharose-4B gel chromatography, octanoicacid-am monium sulfate precipitation, MBP affinity chromatography and proteinA affinity chromatography, respectively. SDS-PAGE showed that serun Ig of tilapia was successfully isolated by protein A affinity column, the heavy chains were shown to be approximately76.6kDa in molecular weight and the light chains were28.5kDa. Sepharose-4B gel chromatography, MBP affinity chromatography and octanoic acid-ammonium sulfate precipitation were not fit for purifying serum Ig of tilapia. The purified Ig was concentrated by Nanosep Centrifugal Devices and the concentration was determined by BCA, the amount of purified serum Ig from2mL serum sample by protein A affinity column was85μg, lower than other fish.2. Five strain hybridomas (T7-B7, T5-E2, T8-G3, T9-D4, T3-D9) against purified serum immunoglobulins of tilapia were developed. T3-D9recognised linear epitope and was specific for the Ig heavy chain, did not show any cross reaction with the serum of Silurus soldatovi meridionalis Chen, Myxocyprinus asiaticus, Lateolabrax japonicus and Ictalurus punctatus. Other four mAbs recognized native form of Ig. T8-G3, T7-B7and T9-D4were specific for the Ig light chain:they all had cross reaction with the serum of Lateolabrax japonicus, while T5-E2was unable to react with the reduced Ig. The results of superposable ELISA test suggested that T8-G3and T9-D4were against similar antigen epitope.3. The dynamics characteristic of specific antibody of tilapia against Aeromonas hydrophila was investigated using mAbs prepared in this study. Tilapia were immunized with Aeromonas hydrophila (group A) and mixture of bacteria and adjuvant (group B), respectively. The results of ELISA using T3-D9showed that specific antibodies can be obviously detected in two groups. Although the level of antibody titer of group A was lower than that group B, the variation pattern was same in two groups. At the21d, titers of group A and B were1:500and1:8000, respectively, but both of them dropped to a half at the28d. After booster immunization at the28d, the titers of the two groups rose obviously, group B was up to1;16000at the49d and group A was1:500at the42d. The titer of groupA reached1:2000at the55d after immunization of Aeromonas hydrophila mixed with adjuvant at42d.In this study, four different isolating methods were applied to find the best way to purify serum Ig from tilapia, which provided basis data for purifing of serum Ig. mAbs against tilapia Ig developed in this study providing a useful immunological tool in monitoring immune response of tilapia and understanding of teleost immune system. |
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