Expression Of Infectious Canine Hepatitis Virus Hexon Loop Gene And Preparation Of Its Monoclonal Antibodies

Objective: Infectious canine hepatitis virus (ICHV) is the causative agent of infectious canine hepatitis (ICH) that is a common acute infectious disease. The mainly host for this virus is the puppy of not more than one years old. Mortality has been reaching to 40%. ICH has spread throughout the world and has become one of the most economically important diseases of the canine cultivation. Immunized serum (isotype or allotype) or gamma globulin is used to sick dogs for emergent prevention and treatment. But its efficiency is limited. At present monoclonal antibody with neutralization activity is the most effective way for prophylaxis and treatment of infectious canine disease, like canine distemper disease and canine parvovirus disease. Surprisingly little was known on infectious canine hepatitis monoclonal antibody.Infectious canine hepatitis virus is also called canine adenovirus typeⅠ(CAV-Ⅰ), belongs to Adenoviridae, Mammalian Adenoviruses, is a kind of virus which has the most pathogenicity in discoverable Adenoviruses. Canine adenovirus is a nonenveloped icosahedral capsid structure. The capsid has three principal protein components: the hexon, the penton, and the fiber. Hexon protein has been extensively studied for structure and function, which can cause stronger neutralization responses. Neutralization antigen determinant exist mainly in hexon protein. The neutralization immunization would be absence if the hexon was replaced or mutation. The hexon has a hexagonal pedestal base from which a tower region projects outward into the solvent. The pedestal base consists of the P1 and P2 region. The tower regions are formed by four loops, L1, L2, L3 and L4. The Loop1 is the longest and most complex, folding back on itself several times and projecting furthest into the solvent, providing maximal interaction with the environment. The sequence analysis of fifteen adenovirus hexon proteins showed that the p1 and p2 regions are conserved, and variable regions exist in Loop1 and Loop2. Seven discrete HVR exist in Loop1 and Loop2. HVR 1 to 6 are found in Loop1, and HVR7 is found at the tip of Loop2. Epitope generating neutralizing antibody are within HVR1, HVR2, HVR4, HVR5 and HVR7.In previous reaseach, two pairs primers were designed and synthesized according to the reported complete nucleotide sequence of canine adenovirus typeⅠin GenBank. The gene of Loop1 and Loop2 were amplified by PCR from ICHV genome obtained from infected dog feces. The two fragments were ligated each other and inserted into vector pET28a (named as pET28aLoop). In this study the plasmid pET28aLoop was transformed into E.coli BL21(DE3), and the recombinant Loop protein was induced by IPTG and purified by the Ni-NTA affinity chromatography column, then using the purified recombinant protein as immunogens to immunize BALB/c mice. Splenocytes were used to fusion with SP2/0 cells. After ELISA screening and subcloning, stable hybridoma cells lines secreting monoclonal antibodies against ICHV were obtained. Positive hybridoma cells were injected into the abdominal cavity of BALB/c mice, and the ascitic fluid was prepared. Screened the anti-ICHV neutralization monoclonal antibodies.Methods:1. The prokaryotic expression plasmid pET28aLoop was transformed into E.coli BL21(DE3) and selected monoclone. The E.coli. BL21(DE3) transformed with the recombinant vectors were cultured and induced with IPTG to express Loop protein. The expressional product was analyzed by SDS-PAGE electrophoresis and its antigenicity was confirmed by Western blotting with the His-specific McAb. The E.coli. BL21(DE3) harboring the recombinant vector of pET28aLoop was lysated with supersonic wave. The supernatant and sediment sample were checked by SDS-PAGE to analysis the solubility of the expressed product. The result was that the protein was expressed in the form of inclusion body. The inclusion bodies were solubilized in 8M urea-NTA0, the supernatant were collected after centrifugate. The fusion protein was purified by Ni-NTA affinity chromatography column. After that, the purified protein was gently renatured by dialyzation in PBS with 6M, 4M, 2M, 0M urea, and concentrated by PEG6000. 2 The purified Loop accompanied with adjuvant were used as immunogen to immunize BALB/c mice for 3 times. The immune interval was two weeks. The titer of antiserum was tested by indirect ELISA used ICHV as the coated antigen. And the mouse was attacked by Loop protein three days before the hybridization. The spleen cells of the immunized mice were hybridized with the myeloma cell SP2/0 by 50% PEG4000. The hybridoma cells were selected by HAT culture medium. The positive hybridoma cells secreting the antibody against Loop were primary detected by the indirect ELISA. Thirty-five hybridomas producing monoclonal antibodies against ICHV were obtained by limiting dilution after 3 times subcloning. Positive hybridoma cell line cells were injected into the abdominal cavity of BALB/c mice. Every mouse was injected with 2×106 cells and the monoclonal antibody ascite was prepared.3 Characterization of monoclonal antibody: The titers of monoclonal antibody ascites and supernament were measured by indirect ELISA used ICHV as coated antigen. Karyotype analysis of hybridoma cell. Hemagglutination inhibition test and neutralization antibody test assays the titer of hemagglutination inhibition and neutralization of monoclonal antibody. The specificity of monoclonal antibody was analyzed by Western blotting. ELISA additivity test was employed to detect whether the corresponding monoclonal antibody recognize different epitype on ICHV. The ascites was purified by the method of caprylic acid-ammonium sulfate precipitation. The characterization of monoclonal antibodies was performed by SDS-PAGE.Results:1 The recombinant plasmid pET28aLoop was transformed into E.coli BL21(DE3) and induced by IPTG. SDS-PAGE analysis showed an induced product band about 36kDa;2 Western blot analysis indicated that the recombinant protein could be recognized by His-specific McAb;3 SDS-PAGE showed that the 36kDa recombinant protein most existed in the precipitation of the lysed bacteria broken by ultrasonic wave, only a little of them was soluble protein;4 The purity of Loop protein was about 95% after purification with Ni-NTA affinity chromatography column;5 The mouse was immunized successfully using recombinant Loop protein as antigen, the serum titer tested by indirect ELISA was up to 1:1.28×103;6 After 2 times of cell hybridation, 201 wells in 960 grow hybridoma cell, the fusion rate was 21%. 2 wells of antibody-secreting hybridoma cells against ICHV were obtained by HAT selection and antibody detection. 35 hybridoma cell lines can secret the McAb against ICHV continuously after coloning, the positive cell strain were subcultured and frozen;7 The titer of the specific antibody in supernatant and ascites were found by ELISA to be 1:1.28×103 and 1:104 respectively. 8 The chromosomal numbers of hybridoma cells were 92～100;9 Four hybridoma cell lines were named 2-7F9, 2-1B1, 7G3 and 8E6, of which monoclonal antibody 2-7F9 and 2-1B1 showed hemagglutination inhibiting activity and titers of its hemagglutination inhibiting were 1:640 and 1:320 respectively.10 Monoclonal antibody 2-7F9 and 2-1B1 had the neutralization activity against ICHV, the titer were 1:640 and 1:320 respectively. Monoclonal antibody 1-7G3 and 1-8E6 haven't.11 Epitope analysis showed that monoclonal antibody of 2-7F9 and 2-1B1 recognize different antigenic epitope.12 Western blotting demonstrated that McAb could specifically combine with a single protein band with molecular weights of 36kDa of recombiant Loop protein and 101kDa of hexon protein of ICHV. There is no positive band in control.13 Ascites purification: 1ml Ascites were used to purify, SDS-PAGE analysis that two bands of antibody were seen under reducing conditions. One is the light chain with molecular weights of 25kDa, the other is the heavy chain with molecular weights of 50kDa. There were also two bands in addition, one lies in between the bands 97.4kDa and 66.2kDa, the other lies in greater than 97.4kDa. But there are more bands in the unpurified Ascites.Conclusions:1 In present study, the recombinant Loop protein was successfully induced, expressed, purified and identificated, which provide valuable materials for further studying the gene of Loop1 and Loop2 of ICHV and the encoding protein function. At the same time it laid a good foundation to construct new genetic engineering product of infectious canine hepatitis.2 After immune BALB/c mice using the recombinant Loop protein, serum with high titer against ICHV was generated, this indicated that the recombinant Loop protein possesses a high immunogenicity.3 Two hybridoma cell lines with neutralization activity secreting monoclonal antibody against ICHV were successfully obtained, it might lay a foundation for further utilizing it to treat the disease and applying it to construct diagnostic method, preparing protein vaccine of infectious canine hepatitis.