CLONING AND EXPRESSION OF A SINGLE-CHAIN VARIABLE FRAGMENT AGAINST THE HEAT-STABLE TOXIN (ST) OF ETEC

AC06

Ozaki, CY (1); Ramos, OHP (2); Elias, WP (1); Piazza, RMF (1).

(1) Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brasil; (2) Biominera, Cientistas Associados, São Carlos, Brasil.

Introduction: Enterotoxigenic Escherichia coli (ETEC) is responsible for at least 400 million acute diarrhea episodes and 700,000 childhood deaths per year. Human ETEC pathogenic strains produce heat-labile toxin (LT) and/or heat-stable toxin (ST) that differ in their structure and function while both are used as markers for detection of infections. When compared to other methods, immunoserological assays present some advantages including high specificity and sensitivity with convenient procedures for sample preparation and assay execution. The advances on antibody biotechnology provide alternatives to obtain low cost antibodies with desirable affinities and specificities by manipulating immunoglobulin domains. One approach consists in cloning immunoglobulin’s heavy and light variable domains (HV and LV) as a single-chain fusion interspaced by a flexible linker, therefore allowing the correct interaction between the domains and preserving the antigen-binding site. Objectives: Construction of a ScFv upon hybridoma cells that produce an anti-ST monoclonal antibody following its bacterial production. Methods: After RNA extraction from hybridoma cells and reverse transcription, coding regions of heavy and light chain variable domains (VH and VL) were PCR-amplified and fused to a linker corresponding to (Gly₄Ser)₃ giving rise to the ScFv-ST coding region. The DNA construct was cloned into p-GEMT Easy vector. The recombinant vector was used as template for sequencing and amplification by specific primers harboring restriction sites (Bam HI and Xho I) intended to permit the subcloning into a pET101D/TOPO vector variant. Aiming to increase the expression and solubility of the target ScFv, this vector was specially designed for its production as a MBP (Maltose Binding Protein) C-terminal fusion. The new recombinant plasmid was used to transform competent E. coli BL21(DE3) cells. Transformed cells were cultured (LB-Amp, 37°C) until reach 0,6 O.D._{600 nm}. After induction of T7 promoter-associated transcription by IPTG (1mM, 3 hours), the cells were harvested, their periplasmic proteins were isolated by osmotic shock and submitted to metal affinity chromatography using Ni-NTA resin and step-wise elution. Fractions were analyzed by SDS-PAGE.  The recognition of ST by purified fractions was tested by western blot. Results: The amplification of VH, VL and ScFv-ST showed fragments containing 375 bp, 352 bp and 851 bp, respectively. Alternative forms of the target protein were identified as two major bands with apparent molecular weight 72 and 45 kDa with no biochemical activity. Discussion: The design of specific primers was necessary once previously performed PCR reactions exhibited low yield. The expression of the transcript as two electrophoretic protein bands could be explained by truncated translation due to the occurrence of rare codons. MBP removal and, consequently, the release of ScFv-ST from its fusion, could probably allow the recombinant protein to manifest its biochemical activities related to ST recognition.