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*

Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.
Humans ; Bacteriophages/genetics* ; Immunoglobulin Variable Region/genetics* ; Gene Library ; Antibodies, Monoclonal/therapeutic use* ; Immunotherapy ; Peptide Library

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

The variable heavy chain region (VH) genes of 3 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL) were cloned and analyzed. The VH family used was VH3-11, VH3-72 and VH3-33. More than 2% difference from the corresponding germline gene was detected in all the 3 obtained potential functional genes (average 16.7). Mutation pattern analysis indicated evidence of antigen selective pressure observed in 1 of 3 cases. Our findings suggested that the tumor cells originate from post-GC cells.
Amino Acid Sequence ; Gene Rearrangement ; Immunoglobulin Variable Region/*genetics ; Leukemia, B-Cell, Chronic/*genetics ; Molecular Sequence Data ; *Mutation

The variable heavy chain region (VH) genes of 3 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL) were cloned and analyzed. The VH family used was VH3-11, VH3-72 and VH3-33. More than 2% difference from the corresponding germline gene was detected in all the 3 obtained potential functional genes (average 16.7). Mutation pattern analysis indicated evidence of antigen selective pressure observed in 1 of 3 cases. Our findings suggested that the tumor cells originate from post-GC cells.
Amino Acid Sequence ; Gene Rearrangement ; Humans ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; Molecular Sequence Data ; Mutation

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

Single domain antibodies against telomerase protein hTERT were prepared by technique of displayed on the surface of recombinant bacterio phages. Total RNA of spleen lymphocytes were extracted from mice immunized recombinant hTERT and transcripted to cDNA. First-strand cDNA was used as a template, heavy chain variable region genes against hTERT were amplified with VHfor and VHback primers by PCR technique. Amplification reaction yielded a fragment about 350 base pairs in length. Amplified cDNA were cloned into the vehide of bacteriophage PCANTAB 5E, the phagemid containing VH gene transformed into competent E. coli TG1, in the presence of helper phage M13K07, VH-g3 fusion proteins were display on the surface of recombinant phages. The phage carrying VH genes that encode binding activities could be detected directly with DOT BLOT. Single domain antibodies were generated successfully and had binding activities with hTERT. Results suggest that phage display technique be a new way of making antibodies. VH genes were cloned successfully, which could provide possibility for futher preparing single-chain antibodies(ScFv) anti-hTERT.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Bacteriophages ; genetics ; Cloning, Molecular ; DNA-Binding Proteins ; Female ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Mice ; Mice, Inbred BALB C ; Telomerase ; immunology

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 evaluate the frequency and mutation status of IgVH gene expression in patients with chronic lymphocytic leukemia (CLL) in China.

METHODSIgVH mutation was detected by multiplex PCR and directly sequencing in 29 CLL patients. IgH somatic hypermutation and mutation site were analysed by IMGT/V-QUEST.

RESULTSOf 29 CLL patients, 21 had IgVH mutation (72%). The most frequently expressed VH gene family was found to be VH3 (55%) followed by VH4 (38%), VH2 (3.5%) and VH7 (3.5%), with no expression of VH1, VH5 and VH6.

CONCLUSIONSThe expression frequency of IgVH gene families in Chinese CLL patients is significantly different from that in Western CLL patients, suggesting the involvement of ethnic and/or environmental factors in CLL development, which might partly explain the different incidence of CLL between China and Western countries.

Adult ; Aged ; Aged, 80 and over ; Chronic Disease ; Female ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; Male ; Middle Aged ; Mutation