Preparation Of Antibody Against ω-CTX MVIIA

Conotoxins (CTXs) are various bioactive polypeptides, which extract from cone snails. They are Conus's main weapon for prey.ω-CTXs are typical N-type voltage sensitive Ca²⁺ channel (VSCC) inhibitor, which can led to limb numbness, paralysis and other symptoms, even seriously to death. So far, there is no simple and effective assay available to detectω-CTXs. In this study, we obtained genetically engineered single-chain antibody against CTX, and established a novel method for detecting CTXs.ω-conotoxins contain 24-29 amino acid. Due to their low molecular weight, they can not induce an adaptive immune response by themselves. So we constructed a fusion protein formed by GST, the Glutathione S-transferase, and CTX to induce an immune response; another fusion protein formed by TRX, the thioredoxin and CTX was used for panning the antibody against the CTX. The GST-CTX concentration was approximately 0.42 mg/mL analyzed by kit and the TRX-CTX concentration was 0.24 mg/mL.After several rounds of immunization, the titers of the antiserum against the CTX were assayed by ELISA. The antibody titer is 32000. Total RNA was extracted from the spleen once the antiserum titer reached a high value. The first strand of cDNA was obtained by reverse transcription which was used as a template for the polymerase chain reaction (PCR) to amplify the heavy and light chain variable domains (V_H and V_L). Subsequently, the amplified V_H and V_L were assembled into single-chain antibody varible fragment (ScFv) using overlap extension PCR by a linker containing a sequence encoding (Gly₄Ser) ₃. Then the ScFv gene was subcloned into pCANTAB-5E to construct a single-chain phage antibody library (4.8×10⁶ cfu/mL). After seven rounds of panning, finally, we obtained scFvs with high affinity and specificity.