Construction, Expression And Identification Of Anti-AFP V_H Single-domain Antibody Fusion Protein Gene

Objective: To construct anti-human AFP V_H Single-domain antibody fusion protein gene,transfect it to E.coliBL-21(DE3) for expression, separate and initial purify anti-human AFP V_H Single-domain antibody fusion protein and identify its bioactivity.Methods: The V_H single domain antibody gene was amplified by PCR from the ScFv vector that has been constrcucted, cloned into pGEM-T vector to constrct recombinant pGEM-V_H vector. pGEM-V_H was transformed into E.coli DH5αand identified. Double digest the pGEM-V_H and TrxA fusion protein express vector pET32a (+) by EcoRâ… and Salâ… , then purify the V_H gene which digested from the pGEM-V_H vector and ligated it into digested pET32a (+) to construct recombinant pET32a-V_H vector. CaCl₂ mediated transformed pET32a-V_H vector into E.coliBL-21(DE3), and identified it by endonuclease digest,PCR and sequencing.The positive clone that was identified was induced by IPTG. Identified the expression product by SDS-PAGE and Western-blot. The positive clone was fermented in a large quantity and collected. Dissolved the germs, separated and purified inclusion. The inclusion lyses denaturalized in 8M urea and dialyzed renaturate in grades density solution to obtain coarse anti-AFP TrxA-V_H single domain antibody fusion protein. We identified the antigen binding ability of the single domain antibody fusion protein by competitive inhibition ELISA with the parent monoclonal antibody,and identified the intemalization by hepatoma carcinoma cell line HepG2 immunocytochemistry. We used the primary hepatic carcinoma pathological section to do immunohistochemistry, normal hepatic tissue as control.Result: (1) The anti-AFP single domain antibody gene was cloned by PCR successfully, and its weight is about 340bp.The result of endonuclease digestion and PCR identification showed that we have constructed the pGEM-T vector successfully. The sequencing result showed that the anti-AFP single domain antibody gene consisted of 339bp.The pET32a-V_H plasma which was transformed into E.coliBL-21(DE3) was expressed at a high level of the total cellular protein in the form of cytorrhyctes. After the fermented bacteriums were dissolved＞washed again and again,denaturized and renaturated, the coase anti-AFP single domain antibody fusion protein was obtained successfully. The result of SDS-PAGE and Western-blot showed that there was the strap of TrxA-V_H fusion protein in the right place, which was about 33KD.(2)The result of competitive inhibition ELISA proved that the expression product has the specific affinity with AFP.(3)The result of hepatoma carcinoma cell line HepG2 immunocytochemistry proved that this product has the better antigen binding and internalization ability. The result of the primary hepatic carcinoma pathological section immunohistochemistry showed that this product also can be applied to diagnose primary hepatic carcinoma.Conclusion: At present, AFP is the most important target antigen, which is used in diagnosis and biotherapy of the primary hepatic carcinoma.The anti-AFP TrxA-V_H single domain antibody fusion protein, whose weight is small, has identified that it has the better ability of antigen binding and internalization by HepG2 immunocytochemistry and the primary hepatic carcinoma pathological section immunohistochemistry. Otherwise, it is a fusion protein, which structure is more stable than ScFv, so it can be used better in the radioimmunoimage and target therapy of the primary hepatic carcinoma probably.