Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.
Animals ; Immunoglobulin Variable Region/genetics* ; Immunoglobulin Fragments/metabolism* ; Single-Chain Antibodies/metabolism* ; Peptide Library ; Mammals/genetics*

This study is (1) to improve the stabilization of human scFv to rabies virus; (2) to prepare active human dsFv fragment; and (3) to evaluate the biological activities of dsFv. The dsFv V(H) and VL were separately expressed in PET22b(+)/BL21 (DE3), solublized and combined in appropriate molar ratio in refolding solution. The resultant dsFv fragments were evaluated for its protection against rabies virus, its affinity and stability, in reference to the cognate scFv. The dsFv was found to bind specifically to Vero vaccine of rabies virus. Compared to the scFv, the dsFv was more stable, had higher affinity, and was able to inhibit the infection of Rabies virus to Vero cell. This established a solid basis for the clinical application of dsFv to rabies virus.
Antibodies, Viral ; immunology ; Disulfides ; chemistry ; Humans ; Immunoglobulin Fragments ; immunology ; metabolism ; Immunoglobulin Variable Region ; immunology ; metabolism ; Rabies virus ; immunology

To develop a fed-batch fementation process of E. coli TOP10 containing a recombinant plasmid pBAD/HBs Fab. Cells were grown in semi-defined medium at 37 degrees C, and the feed operation using glycerol as carbon source was performed when dissolved oxygen increased. When the target cell concentration reached to 64g/L, arabinose was added to a final concentration of 0.02%. Cells were grown for another 5h with the culture temperature decreased from 37 degrees C to 30 degrees C. In the whole process, cell growth was monitored by measuring OD600 of samples taken at 1/2h intervals and the dissolved oxygen was kept above 30%. After the fementation, E. coli pellets were collected for purification of Fab protein. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. Cell concentration we got is 96g wet bacteria per liter, the Fab protein is about 6% of total protein of the host, that is 80mg per liter. Stable fermentation parameters were obtained for fermentation to improve productivity of the Fab protein. The Fab protein was produced in the form of soluble biologically active protein, it's better than inclusion bodies from which biologically active protein can only be recovered by complicated and costly denaturation and refolding process.
Blotting, Western ; Escherichia coli ; genetics ; metabolism ; Fermentation ; physiology ; Hepatitis B Surface Antigens ; immunology ; Immunoglobulin Fab Fragments ; biosynthesis ; genetics ; immunology ; Temperature

To construct the recombinant vector pBV220-scFv and express anti-clenbuterol (CBL) scFv antibody in Escherichia coli, we amplified the scFv gene using plasmid pCANTABSE-CBL as a template, recombined it with pPICZalphaA, then amplified the scFv-His-tag gene from plasmid pPICZalphaA-scFv and linked it with expression plasmid pBV220. We identified the recombinant plasmid by restrictive enzyme digestion, PCR amplification and sequence analysis. Finally, we transformed the recombinant vector into E. coli DH5alpha that was temperature-induced and expressed recombinant protein. We identified the recombinant protein by SDS-PAGE, Western blotting and indirect competitive ELISA. The results show that recombinant plasmid pBV220-scFv contained the inserted fragment with highest homology about 99.8%. The expression of scFv induced by temperature show 37 kD Mw and anti-His-tag mAb recognized-activity by SDS-PAGE and Western blotting respectively, and could competitively combine with CBL, the IC50 is 4.55 ng/mL. The recombinant plasmid pBV220-scFv is constructed and expresses the scFv gene of CBL in E. coli successfully. This study suggests the corresponding immunoassay methods could be set up by the recombinant scFv.
Antibodies ; immunology ; Clenbuterol ; immunology ; Cloning, Molecular ; Genetic Vectors ; genetics ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

AIMTo construct recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/zeta gene and detect its expression in Jurkat cells.

METHODCD28-zetacDNA were amplified from plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. Objective gene expression was determined by PCR and FACS. Jurkat cells were transfected with high titer of retroviruses and resistant clones were obtained by G418 selection. Objective mRNA was determined by RT- PCR.

RESULTSThe recombinant eukaryotic vector was constructed successfully by PCR and enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses by PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. Objective gene was amplified from Jurkat cells transfected with retroviruses by RT-PCR.

CONCLUSIONRecombinant retrovirus expressing anti-CD20 scFv/CD80/CD28/zeta gene was successfully constructed and objective protein could be expressed in Jurkat cells.

Antigens, CD20 ; genetics ; metabolism ; B7-1 Antigen ; genetics ; metabolism ; CD28 Antigens ; genetics ; metabolism ; Genetic Vectors ; Humans ; Immunoglobulin Fab Fragments ; genetics ; metabolism ; Immunoglobulin Variable Region ; genetics ; metabolism ; Jurkat Cells ; Recombinant Fusion Proteins ; genetics ; metabolism ; Retroviridae ; genetics ; T-Lymphocytes ; metabolism ; Transfection

Both interleukin-1 and IgE are important in the pathogenic mechanism of the allergy asthma. cDNA of interleukin-1receptor antagonist (IL-1ra) and IgE were cloned and a prokaryotic expression vector IL-1ra-Fcepsilon/pBV220 was constructed. The vector was transformed into Escherichia coli BL21(DE3). The fusion protein was expressed successfully in the form of inclusion body. The recombination protein of IL-1ra-Fcepsilon was highly purified by chromatography of gel filtration and ion exchange, which was identifited by Western blotting. The cell assay showed that the activity of IL-1ra-Fcepsilon was as high as IL-1ra in vitro after refolding. The pharmacokenetic profile of IL-1ra-Fcepsilon and L-1ra was analyzed, and the half time of IL-1ra-Fcepsilon is 4.78 times than that of IL-1ra.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Fusion ; Immunoglobulin E ; genetics ; metabolism ; Immunoglobulin Fc Fragments ; genetics ; metabolism ; Interleukin 1 Receptor Antagonist Protein ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

Human transferrin receptor (TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b(+) and transformed into E. coli BL21(DE3) . Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells. The scFv was purified by one-step affinity chromatography with Ni+ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.
Animals ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; metabolism ; HeLa Cells ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Transferrin ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; metabolism

We studied the aggregation of a recombinant engineering antibody (chA21). Anti-ErbB2 antibody chA21 was produced by fusing single-chain Fv (scFv) with human IgG1 Fc fragment, and it was proved to be a drug candidate for cancer therapy. We characterized the aggregation of chA21 by high performance sized-exclusive chromatography (HPSEC), dynamic light scattering (DLS), SDS-PAGE, indirect ELISA assay, and compared the influence of temperature and additive on the level of aggregation and binding activity. Conformation changes of different levels of aggregation were also analyzed via circular dichroism (CD). Finally, we analyzed which part of chA21 was involved in aggregation by cleaving it into scFv and Fc fragments. The results showed that chA21 could form aggregates in the storage solution. The aggregates interacted through non-covalent bonds and remained binding activity. Temperature and additive could slightly affect the level of aggregation and binding activity, while the conformations of chA21 were stable. Aggregation propensity of scFv fragment was almost same as chA21, indicating that scFv may be the major part to form the aggregates. The research on aggregation may be helpful to develop a suitable formulation for chA21 clinical application as well as provide direction for future antibody design and reconstruction.
Antibodies ; chemistry ; metabolism ; Humans ; Immunoglobulin Fc Fragments ; chemistry ; metabolism ; Immunoglobulin Variable Region ; chemistry ; metabolism ; Protein Conformation ; Protein Engineering ; methods ; Receptor Aggregation ; immunology ; Receptor, ErbB-2 ; chemistry ; immunology ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; immunology

BACKGROUNDMonoclonal antibodies (mAbs) such as DD3, raised against progastrin-releasing peptide(31-98) (ProGRP (31-98)) antigen, have been used to target small cell lung cancer (SCLC). However, as an intact mAb, DD3 is cleared slowly from the body, with an optimal radioimmunoimaging time of 72 hours. More recently, a single-chain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research. Thereby, it potentially increases the radioimmunoimaging efficacy. However, there have been few studies with this antibody fragment. The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of (131)I-anti-ProGRP(31-98) scFv in nude mice bearing SCLC xenografts.

METHODSAnti-ProGRP(31-98) scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry. (131)I-anti-ProGRP(31-98) scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram (%ID/g) in various organs was calculated. Similarly, the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated. After injection of (131)I-anti-ProGRP(31-98) scFv, treated mice were imaged at 1, 24, and 30 hours. Then the tumor/base ratios were calculated.

RESULTSProGRP was highly expressed in NCI-H446 cells and xenograft tissue. The metabolism of (131)I-anti-ProGRP(31-98) scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes, respectively. The %ID/g of (131)I-anti-ProGRP(31-98) scFv in xenografts was much higher than in healthy tissues at 12 hours after injection, reaching a maximum of (5.38±0.92) %ID/g at 24 hours. Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours, with 24 hours proving optimal.

CONCLUSION(131)I-anti-ProGRP(31-98) scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid blood clearance, indicating that it could be a promising agent for SCLC radioimmunoimaging.

Animals ; Female ; Flow Cytometry ; Humans ; Immunoglobulin Fragments ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Peptide Fragments ; immunology ; Radioimmunodetection ; methods ; Recombinant Proteins ; immunology ; Small Cell Lung Carcinoma ; diagnostic imaging ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli.

METHODSThe specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test.

RESULTSIt was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1.

CONCLUSIONVH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.

Adenocarcinoma ; metabolism ; pathology ; Antibodies, Monoclonal ; genetics ; immunology ; metabolism ; Carcinoma, Small Cell ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; metabolism ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B ; immunology ; Humans ; Immunoglobulin Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lung Neoplasms ; metabolism ; pathology