Construction and characterization of VL–VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins
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 VL–VH 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 (VL) and heavy chain (VH) 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 VH–VL tail-parallel co-expression vectors based on the “5′-VH-ivs-IRES-VL-3′” mode, the ivs-IRES fragment and VL genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and VH 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 VL–VH 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 VL–VH tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.
KeywordsStaphylococcal enterotoxins Germ-line genes, variable region Genetically engineered recombinant antibodies Disulfide stable Self-assembly
This work was supported by the National Natural Science Foundation of China (No. 31272540) and Tianjin science and technology support key project plan (13ZCZDNC08800) in China.
Conflict of interest
The author (s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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