Generation Of Single-chain Fv Antibody Against Epitope PreS1(20-47) Of HBV And Its Expression In E.coli And Tobacco

PreSl (20-47) is one of the important epitope of HbsAg, which has been pointed out to have relationship with infection of HBV. Humanized antibody against PreSl with high affinity is a new way to block HBV. In addition, the display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities. Here we explored the use of this technology to find human antibodies with biological properties. A phage-scFv specific for PreS 1 (20-47), with an affinity of 1x10-7 -1x10-8M, was isolated from a phage display immune library, which was constructed with PBLs immunized by PreSl conjugated to BSA in vitro. Sequencing of the scFv showed that heavy chain belongs to VH4 and light chain belongs to V A 4 subgroup respectively. This study obtained the high affinity human scFv against PreS 1(20-21) for the first time. It is the foundation of therapy of Hepatitis B.The gene of scFv was cloned into the expression vector pET32a(+) and subsequently expressed in Escherichia coll. The expressed scFv fused with the 109 aa Trx-Tag thioredoxin protein was above 30% total cell protein in the shaking flask cultures. After dissolving inclusion bodies, renaturing inclusion bodies and further purification by immobilized metal ion affinity chromatography (IMAC), the fusion protein Trx-scFv of electrophoretic purity was obtained, they still possessed antigen-binding activity.Plants offer various advantages for the production of pharmaceutical proteins over conventional production systems such bacterial or mammalian cell culture. In order to explore transgenic plants for production of recombinant scFv specific for PreSl (20-47), Nicotiana tabacum was transformed with a gene encoding anti-PreSl of hepatitis B surface antigen scFv and bearing an //-terminal endoplasmic reticulum protein signal peptide sequence. High accumulation of the scFv protein in transgenic tobacco plants was achieved by retention of the recombinant antibodies in the lumen of the endoplasmic reticulum (ER). Expression levels of scFv antibodies reached up to 0.5% of total soluble proteins in leaves. The biologically active scFv was easily purified (to 90% purity) from SAFH4 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the SAFH4 plant line indicates that 9 (JL g of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.The production of the scFv antibody proteins in plant root exudates was also addressed. The scFv antibody protein was continuously secreted from the transgenic tobacco roots into a simple hydroponic medium at 630 to 760 ng g-1 dry weight of root day-1. The antibody was about 2% of the total secreted protein and still possessed antigen-binding activity. It is a potential way to product recombinant scFv proteins.