title: Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.
abstract: Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.
authors.affiliation: School of Life Science, Tianjin University, No. 92, Weijin Road, Nankai District, Tianjin, 300072, China.
authors.forename: Xianzhi
authors.initials: X
authors.lastname: He
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Pengchong
authors.initials: P
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Deng
authors.forename: Jinhai
authors.initials: J
authors.lastname: Huang
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000008:administration & dosage*; D002460:Cell Line; D006224:Cricetinae; D004768:Enterotoxins / Q000276:immunology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007167:Immunotherapy / Q000379:methods*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000502:physiology*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000008:administration & dosage*; D013202:Staphylococcal Food Poisoning / Q000276:immunology / Q000628:therapy*; D013203:Staphylococcal Infections / Q000276:immunology*; D013211:Staphylococcus aureus / Q000502:physiology*; D034421:Sus scrofa
chemical_list: D000907:Antibodies, Bacterial; D004768:Enterotoxins; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins