OBJECTIVETo construct a human phage display antibody library, which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic. This can provide a platform for human antibody preparation and diagnosis, prophylaxis and therapy of RSV infection in children.

METHODSPeripheral blood lymphocytes were isolated from 52 children with RSV infection. cDNA was synthesized from the total RNA of lymphocytes. The light and heavy chain Fd (VH-CH1) fragments of immunoglobulin gene were amplified by RT-PCR. The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue. The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs. The plasmids extracted from amplified E.coli were digested with restriction endonucleases Sac I, Xba I, Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes. RSV virions were utilized as antigens to screen Fab antibodies.

RESULTSBy recombination of light and heavy chain genes, an immune Fab phage display antibody library against RSV containing 2.08x10(7) different clones was constructed, in which 70% clones had light chains and heavy chain Fd genes. The capacity of Fab phage antibody gene library was 1.46x10(7) and the titre of the original Fab antibody library was about 1.06x10(12) pfu/mL. The antibody library gained an enrichment in different degrees after the preliminary panning.

CONCLUSIONSUtilizing the technology of phage display, an immune Fab phage display antibody library against RSV was successfully constructed in this study, which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis, therapy and prophylaxis of RSV infection in children.

Antibodies, Viral ; genetics ; Bacteriophages ; genetics ; Child ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Peptide Library ; Respiratory Syncytial Viruses ; immunology

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*

There are great differences in the clinical process and prognosis of the chronic lymphocytic leukemia(CLL), the precise diagnosis is of importance to judge prognosis, guide therapy and research pathogenesis mechanism. The variable region mutation of immunoglobulin heavy chain is the most stable molecular index of disease prediction. The patients with sequence of IgHV somatic high mutation usually have a better prognosis and a more longer survival time than those without the mutation. The recent study has found that specific IgHV gene expression also can predict the disease outcome in some cases regardless of mutation. The clinical prognosis of CLL patients can be further stratified by specific IgHV gene expression. In this review, the progress in the research of the clinical significance of specific IgHV gene expression in the chronic lymphocytic leukemia is summarized.
Humans ; Immunoglobulin Variable Region ; genetics ; Immunoglobulin gamma-Chains ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; genetics ; Mutation ; Prognosis

Functional heavy-chain antibodies (HCAbs) lacking light chains occur naturally in camels. The variable domain of heavy chain of heavy-chain antibody is referred to VHH. The VHH gene family is homologous to human VH subgroup III. The single-domain VHH antibodies are constructed by cloning the variable domains of HCAbs. Compared to human VHs, VHH germ-line sequences contain some hallmark substitutions in framework region 2, including V37F(Y), G44 E, L45 R, W47G. The substitutions at positions 44, 45, 47 are often used to camelise the human VHs. Being a small binders, VHH antibodies are well expressed, extremely stable and very soluble. Camelised human VHs are proved to exhibit the same qualities as those of VHH antibodies. The single-domain VHH antibodies will be useful in the drug development and basic research.
Animals ; Binding Sites, Antibody ; Camelus ; immunology ; metabolism ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Objective: To investigate the mutational features of the immunoglobulin heavy chain variable region (IgHV) gene in patients with chronic lymphocytic leukemia (CLL) using immunophenotypic and molecular genetic methods. Methods: The laboratory results of 266 CLL patients who underwent IgHV gene examination at Sino-US diagnostics laboratory from February 2020 to February 2021 were analyzed for the IgVH mutational status and presence of specific IgVH fragments. In addition, their immunophenotypic, molecular, chromosomal karyotypic, and FISH profiles were investigated and correlated with the IgVH mutational status. Results: Among 266 patients, 172 were male and 94 were female, with a media age of 67 years (20-82 years).There were more patients with mutated IgHV (m-IgHV) than unmutated IgHV (un-IgHV) (69.2%∶30.8%). There was association of VH family and the presence of gene fragments: the overall incidence of VH families including VH3 family (142/266, 53.4%), VH4 family (75/266, 28.2%), and VH1 family (34/266, 12.8%) was about 95%, among which the proportion of VH4-34 (26/266, 9.8%), VH3-23 (25/266, 9.4%), VH3-7 (24/266, 9.0%), and VH4-39 (16/266, 6.0%) was about 35%. VH3-20 and VH3-49 only occurred in un-IgHV (P<0.05). In addition, the expression rates of CD38 (26.3% vs. 3.0%), CD79b (71.1%∶45.5%) and 11q deletion (25.5%∶5.3%) were higher in un-IgHV, and single trisomy 12 (37.9%∶5.6%) were more commonly found in m-IgHV (P<0.05). MYD88 was one of the major mutation genes in m-IgHV, while ATM had the highest mutation rate in un-IgHV. Conclusion: CLL patients have differential expression in terms of IgHV gene mutations, correlating to their immunophenotype and genetics characteristics.
Male ; Female ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/genetics* ; Immunoglobulin Variable Region/genetics* ; Genes, Immunoglobulin Heavy Chain ; Mutation ; Immunoglobulin Heavy Chains/genetics* ; Prognosis

Immunoglobulin A vasculitis (IgAV), also known as Henoch-Schönlein purpura, has complex etiology and pathogenesis which have not been fully clarified. The latest research shows that SARS-CoV-2 and related vaccines, human papilloma vaccine, and certain biological agents can also induce IgAV. Most studies believe that the formation of galactose-deficient IgA1 (Gd-IgA1) and Gd-IgA1-containing immune complex plays a crucial role in the pathogenesis of IgAV. It is hypothesized that the pathogenesis of IgAV is associated with the binding of IgA1 to anti-endothelial cell antibodies. In addition, genetics also constitutes a major focus of IgAV research. This article reviews the new advances in the etiology of IgAV and summarizes the role of Gd-IgA1, Gd-IgA1-containing immune complex, anti-endothelial antibody, IgA1 conjugates, T lymphocyte immunity, and genetic factors in the pathogenesis of IgAV.
Humans ; IgA Vasculitis ; Antigen-Antibody Complex ; Immunoglobulin A/genetics*

To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system. The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood. The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF' with the Hte gene. Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times. The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations. The library was calculated to have a diversity of 1.7 x 10(11) and validated by the selection of antibodies against six different protein antigens. This library provides the basis for further selection of antibody-based drugs. It is the first time to report that XL2-blue MRF' can be used to improve the diversity of the library in the recombination system.
Adult ; Escherichia coli ; genetics ; immunology ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Infant, Newborn ; Integrases ; metabolism ; Lymphocytes ; immunology ; Peptide Library ; Recombination, Genetic ; genetics ; Single-Chain Antibodies ; genetics ; metabolism ; Transformation, Genetic

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

OBJECTIVETo construct a personalized full-length fully human antibody mammalian display library for children with systemic lupus erythematosus (SLE).

METHODSThe total RNA was isolated from the PBMCs of SLE children. The heavy chain variable region and kappa light chain (VH and LCκ) of the antibody genes were amplified by RT-PCR and inserted into the pDGB-HC-TM vector separately to construct the heavy chain and light chain libraries. The library DNAs were transfected into 293T cells and the expression of full-length fully human antibody on the surface of 293T cells was analyzed by flow cytometry.

RESULTSUsing 0.8 µg total RNA as the template, the VH and LCκ were amplified and the full-length fully human antibody mammalian display library was constructed. The VH and LCκ gene libraries had a size of 9.4×10(4) and 8.4×10(4), respectively. Sequence analysis of 10 clones randomly selected from the VH and LCκ gene libraries each showed that 8 heavy chain clones and 7 light chain clones contained correct open reading frames, and flow cytometry demonstrated that all the 15 clones express full-length antibodies on 293T cell surfaces. 293T cells co-transfected with the VH and LCκ gene libraries expressed the full-length antibodies on the cell surface.

CONCLUSIONThe personalized full-length fully human antibody library for SLE children constructed allows display of the full-length antibodies on mammalian cell surfaces, thus providing a valuable platform for analyzing the autoantibodies, their etiological role, and their clinical implications in SLE.

Amino Acid Sequence ; Child ; Gene Library ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Lupus Erythematosus, Systemic ; genetics ; immunology ; Membrane Proteins ; genetics

The DNA coding for the fusion protein of thromobopoietin mimetic peptide (TMP) and human IgG1 Fc fragment was amplified from recombinant plasmid pET28a/TMPFc, inserted into pPICZalphaA and transformed into Pichia pastoris using electroporation. The recombinants of correct phenotype were identified after screening on MDH and MMH culture medium. The fusion gene was verified with PCR and western blot. MTT method was used to test the activity of TMPFc in promoting the growth of Ba/ F3-mpl cell. The TMPFc with a 64 000 molecular weight was a secretary protein in the system and its expression amounted to 65% of the total protein in the medium supernatant. The TMPFc showed a promotive effect on the growth of Ba/F3-mpl in vitro. A significant portion of the secretary protein existed as dimer, which provided material for studying the dimer in future.
Amino Acid Sequence ; Blotting, Western ; Humans ; Immunoglobulin Fc Fragments ; genetics ; Immunoglobulin G ; genetics ; Molecular Sequence Data ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; Thrombopoietin ; genetics