Cloning Of The Variable Region Of Anti-anti-idiotypic Monoclonal Antibody NP48 Of Schistosoma Japonicum And Construction, Expression And Functional Analysis Of NP48's Recombinant Monospecific Bivalent Diabodies

Guan Xiaohong,et al.(1991)established a cell line of the monoclonal anti-idiotypic antibody (anti-id) NP30 of Schistosoma japonicum, which was testified to be internal image of gut associated antigen (GAA) of schistosomula. They found that NP30 had partial cross-reaction with soluble egg antigen (SEA) and membrane associated antigen (MAA). Not only NP30 could be used as "antigen reagent" in the immunodiagnostic assays based on antibody detection of schistosomiasis japonica, but also NP30 had good efficacies of anti-pathology immunity and anti-infection protective immunity to schistosomiasis japonica. For separating and purifying NP30 and the antigen molecular simulated by NP30, the monoclonal anti-anti-idiotypic antibody (anti-anti-id, Ab3) of NP30 were obtained by fusion of myeloma cells SP2/0 and spleen cells from BALB/c mice immunized with HRP-NP30, named NP48. The isotype of NP48 was determined as IgG2b. The McAb NP48 reacted positively to SEA and SWAP respectively when detected with ELISA (Enzyme-Linked Immunosorbnent Assay). For broadening the utilization area of NP48 and for construction, expression the bispecific diabodies which will be use to diagnose and cure schistosomiasis japonica, our studies were designed to clone the variable region genes of light and heavy chains (VL and VH) ofmonoclonal anti-anti-idiotypic antibody NP48 of Schistosoma japonicum and construction, expression and characterization of a single-chain monospecific diabody of NP48 based on the studies abroad and domestic on scFv multimer.Objectives:1. To clone the variable region genes of light and heavy chains (VL and VH) of monoclonal anti-anti-idiotypic antibody NP48 of Schistosoma japonicum, and to analyze the nucleotide sequences of the VL and VH genes.2. To construct a single-chain diabody(scFv dimer) gene derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum and to express and characterize the protein.Methods and Results:1. The hybridoma cells secreting monoclonal anti-anti-idiotypic antibody NP48 of Schistosoma japonicum were cultured and their total RNA was extracted. The desired VL and VH fragments were amplified by RT-PCR from the total RNA. The PCR products were then cloned into pMD18-T vector and sequenced. The VL and VH gene were compared with GenBank and published mouse variable region genes.It was proved that the full-length VL gene was 336bp,and a member of mouse Ig chain subgroup II and originated from rearrangement of germ line he24 and JK4 genes. The VL gene sequence was registered by GenBank (accession No.AY309010). It was proved that the full-length VH gene was 354bp,and a member of mouse Ig heavy chain subgroup II and originated from rearrangement of germ line J558.47 , Dsp2.2 andJH4 genes. The VH gene sequence was registered by GenBank (accession No. AY312433).2. The monospecific diabody genes were constructed by SOE(splicing by overlap extension) and using Gly4Ser as a linker to join the C-terminus of the VH to the N-terminus of the VL/ VP(VP is the part of the nucleotide sequence of pseudogene from the light chain). And then the genes were subcloned into pMD18-T vector for DNA sequencing analysis. Target DNA fragments in pMD18-T-DR/ Dp were digested respectively with restriction enzymes,and then linked respectively with arabinose-inducible vector, pBAD/gIII. There were less soluble target proteins in the supernates and higher target proteins in the pellets as inclusion body when separating the DR expression proteins. And the insoluble fraction was recovered as a soluble ,correctly processed protein by solubilising with 8M urea. Use the HiTrap affinity columns to purify the (His)6-tagged recombinant protein expressed as inclusion body. The antigen binding activity of DR expressed product was detected with Western-blot and Dot-ELISA.The VH-G4S-VL gene was confirmed by sequencing. The recombinant were determined by digesting with endonucleases and expected bands were identified. The specificity of the expressed protein with poly-His6 tag was confirmed by West...