title: [Construction and expression of human-mouse chimeric antibody against human CD40].
abstract: OBJECTIVE
  To construct and express a chimeric antibody against human CD40 molecule by genetic engineering antibody transformation.
  
  
  METHODS
  Total RNA was extracted from the murine hybridoma cell 5C11 which secreted anti-CD40 monoclonal antibody (mAb). The genes encoding V(H) and V(L) of mAb 5C11 were amplified by RT-PCR. According to sequence analysis, the primer was designed to amplify signal peptide sequences relative to V(H) and V(L) genes. The V(H) and V(L) genes of mAb 5C11 and relative signal peptide sequences were spliced with C(H) and C(kappa) genes of human IgG1 to construct expression plasmid pIRES/h5C11 of human-mouse chimeric antibody gene and the plasmid was transfected into 293T cells under Lipofectamine mediation for transient expression. Expressed product was analyzed by flow cytometry.
  
  
  RESULTS
  The result of NCBI gene data bank blast revealed that cloned gene sequence accorded with mice' V(H) and V(L) genes and feature of signal peptide sequence. The constructed plasmid pIRES/h5C11 was correct. The restriction endonuclease digestion analysis and PCR showed that the recombinant genes were subsequently cloned into vector pIRES. FACS analysis showed that human-mouse chimeric antibody against human CD40 maintained the binding activity and specificity to human CD40 molecule.
  
  
  CONCLUSIONS
  The gene encoding variable region of human-mouse chimeric antibody against CD40 is cloned successfully and the human-mouse chimeric antibody against CD40 is expressed transiently in 293T cells.
authors.affiliation: Institute of Medical Biotechnology, Key Laboratory of Medicine and Clinical Immunology of Jiangsu Province, Soochow University, Suzhou 215007, China.
authors.forename: Qiu-xia
authors.initials: QX
authors.lastname: Qu
authors.forename: Yong-jing
authors.initials: YJ
authors.lastname: Chen
authors.forename: Yan
authors.initials: Y
authors.lastname: Ge
authors.forename: Qin
authors.initials: Q
authors.lastname: Wang
authors.forename: Cheng
authors.initials: C
authors.lastname: Chen
authors.forename: Ming-feng
authors.initials: MF
authors.lastname: Yang
authors.forename: Xue-guang
authors.initials: XG
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D019013:CD40 Antigens / Q000276:immunology* / Q000378:metabolism*; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000276:immunology* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins