Production And Application Of Monoclonal Anti-idiotypic Antibody Of WSSV Envelope Protein VP28

White spot disease (WSD) which is caused by white spot syndrome virus (WSSV) is one of the most important lethal diseases that affect current shrimp aquaculture. During the past more than ten years since WSD outbreak in 1993, many papers on researches of pathogen, pathology, detecting and diagnostic methods of the disease have been published. Recent works focus mainly on figuring out the infection mechanism and searching the strategies of prophylaxis and control of WSSV infection. WSSV-VP28 has been proved to play a very important role in WSSV infection process.In this thesis, anti-VP28 monoclonal antibody (Mab) 1D6 (Ab1), which was proved to specifically recognize VP28 and be able to neutralize WSSV infection in vivo and vitro previously, was purified and used as antigen immunized to BALB/c mice, in order to generate monoclonal anti-idiotypic antibodies (Ab2). After cell fusion, monoclonal antibody A1A5 was obtained after screening by indirect enzyme-linked immunosorbent assay (ELISA) and competitive ELISA. Mab A1A5 was confirmed to be able to inhibit the binding between rabbit anti-VP28 antibodies and WSSV significantly. Anti-A1A5 antibodis (Ab3) were generated and used to further analyze the characterization of Mab A1A5, and Mab A1A5 was proved to represent the internal image of VP28 exactly. Mab A1A5 was used to study the interaction between VP28 and host cells, detect the combination of VP28 with Chinese shrimp (Fenneropenaeus chinensis) tissues, identify the VP28 binding protein on haemocyte membrane of Chinese shrimp. This study shed new light on investigating the role of VP28 in WSSV infection process, provided new information for in-depth study of WSSV infection mechanism and searching the strategies of prophylaxis and control of WSSV infection. The details are as follows:(1) Production of monoclonal anti-idiotypic antibody of VP28. Anti-VP28 Mab 1D6, which was proved to specifically recognize VP28 and be able to neutralize WSSV infection in vivo and vitro previously, was produced in ascites, purified by caprylic acid-ammonium sulfate (CA-AS) and Protein G agrose. Purified Ab1 was immunized to BALB/c mice as antigen, in order to generate monoclonal anti-idiotypic antibodies. After cell fusion, Mab A1A5 which was confirmed to be able to inhibit the binding between rabbit anti-VP28 antibody and WSSV significantly was identified by indirect ELISA and competitive ELISA.(2) Using anti-AlA5 antibodies to further analyze the characterization of Mab A1A5. Mab A1A5 was purified and immunized to BALB/c mice as antigen to generate anti-A1A5 antibody (Ab3), in order to verify whether Mab A1A5 could represent the internal image of VP28 exactly. Competitive ELISA, indirect immunofluorescence assay (IIFA), immunogold electron microscopy (IEM) and in vivo neutralizing assay were developed to analyze Ab3. Competitive ELISA showed that Ab3 could dose-dependently prevent the binding between WSSV and rabbit anti-VP28 antibody. Furthermore, IIFA, IEM and in vivo neutralizing assay showed that both Abl and Ab3 which was induced by Mab A1A5 could combine with WSSV envelope and reduce WSSV infection, Ab3 recognized the same antigen epitope with Ab1. According to the theory of anti-idiotypic antibodies, all the results indicated that the monoclonal anti-idiotypic antibody of VP28 which represented an internal image of VP28 was successfully developed.(3) Application of monoclonal anti-idiotypic antibody of VP28. The monoclonal anti-idiotypic antibody of VP28 was used to study the interaction between VP28 and host cells. IIFA confirmed that Mab A1A5 could combine with the haemocyte membrane of Chinese shrimp, it indicated that there was VP28 receptor protein on the haemocyte membrane of Chinese shrimp. Furthermore, anti-WSSV Mabs were labeled with horse radish peroxidase (HRP) and used in vitro WSSV blocking experiments of Dot-blot and ELISA. Haemocyte membrane of Chinese shrimp was dotted on NC membrane or coated on ELISA plate, after incubated with Mab A1A5 or Mab 1G8 (anti-haemocyte granules Mab), then incubated with WSSV. The binding of WSSV was detected with anti-WSSV-HRP. The blocking experiments showed that the Mab A1A5 could block the binding of WSSV with haemocyte membrane. It provided a new basis to prove that VP28 played a key role in WSSV infection process. IIFA and immunohischemical (IHC) were developed to indentify whether Mab A1A5 could combine with gill, intestine, hepatopancreas, muscle, heart and gonad of Chinese shrimp, both of the results showed that Mab A1A5 could react with gill and intestine, but did not react with hepatopancreas, muscle, heart and gonad. It demonstrated that there was VP28 receptor protein in gill and intestine of Chinese shrimp. Coimmunoprecipitation was developed to identify the VP28 binding protein on haemocyte membrane of Chinese shrimp. A 47 kDa protein which was specially recognized by Mab A1A5 was identified by coimmunoprecipitation, and it was considered to be VP28 receptor protein on Chinese shrimp haemocyte membrane.