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

OBJECTIVE: To analyze the correlation between the mutational status of immunoglobulin heavy chain variable (IGHV) gene with the prognosis of patients with Waldenström macroglobulinemia (WM). METHODS: Immunoglobulin heavy chain gene (IGH) clonotypic sequence analysis was carried out to assess the mutational status of IGHV in the blood and/or bone marrow samples from 44 WM patients. The usage characteristics of IGHV-IGHD-IGHJ gene was explored. RESULTS: The most common IGHV subgroup was IGHV3, which was similar to the data from the Institute of Hematology of Chinese Academy of Medical Science. IGHV3-23 (20.45% vs. 15.44%) and IGHV3-74 (11.36% vs. 7.35%) were the main fragments used, which was followed by IGHV4 gene family (15.91% vs. 24.26%). However, no significant correlation was found between the IGHV4 usage and the prognosis of the patients. Should 98% be taken as the cut-off value for the IGHV mutation status, only 5 patients had no IGHV variant, and there was no correlation with the prognosis. Based on the X-tile analysis, 92.6% was re-selected as the cut-off value for the IGHV variant status in such patients. LDH was increased in 26 patients (59.1%) without IGHV variant (P < 0.05), whilst progression-free survival (P < 0.05) and overall survival (P < 0.05) were significantly shorter compared with those with IGHV variants. CONCLUSION The usage characteristics of IGHV-IGHD-IGHJ in our patients was similar to reported by the Institute of Hematology of Chinese Academy of Medical Science, albeit that no correlation was found between the IGHV4 usage and the prognosis of the patients. Furthermore, 98% may not be appropriate for distinguishing the IGHV variant status in WM patients.
Humans ; Immunoglobulin Heavy Chains/genetics* ; Multigene Family ; Mutation ; Waldenstrom Macroglobulinemia/genetics*

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

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

OBJECTIVETo construct full-length human bladder cancer-specific antibody libraries for efficient display of full-length antibodies on the surface of mammalian cells.

METHODSThe total RNA was isolated from peripheral blood mononuclear cells from patients with bladder cancer. The repertoires of IgG1 heavy chain variable region (VH) and Kappa light chain were amplified by RT-PCR using specific primers. The antibody genes were inserted into the vector pDGB-HC-TM to construct the bladder-cancer-specific antibody libraries of heavy chains and light chains. Ten clones from each library were randomly picked for gene sequencing and transient transfection into FCHO cells to analyze antibody display on mammalian cell surface by flow cytometry after staining with corresponding fluorescent labeled antibodies.

RESULTSThe libraries of bladder-cancer-specific antibody heavy chain (IgG1) and light chain (LCk) were successfully constructed. Seven out of the 10 clones randomly selected from the heavy chain library and 9 out of the 10 clones from the light chain library showed correct open reading frame, coding for 7 unique VH and 9 unique LCk. The combinatory library size reached 3.32×10(11).

CONCLUSIONWe have successfully constructed a full-length human bladder-cancer-specific antibody library with a combinatory diversity of 3.32×10(11) based on mammalian display technology, which can be used for screening monoclonal antibodies against bladder-cancer-associated antigens.

Amino Acid Sequence ; Animals ; Antibodies ; genetics ; Cell Surface Display Techniques ; Gene Library ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Peptide Library ; Urinary Bladder Neoplasms ; genetics ; immunology

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

To investigate the optimal reaction conditions of clonal rearrangements of immunoglobulin heavy chain (IgH) gene using polymerase chain reaction (PCR) with consensus primers and the feasibility of detecting this gene using SYBR green I real-time quantitative PCR with consensus primers, the systemic experiments were performed, which included annealing temperature, concentration of primer, the amount of Taq enzyme, concentration of dNTP and Mg(2+), number of PCR cycles. Detection of this gene on SYBR green I real-time quantitative PCR with consensus primers was carried out under the optimal reaction parameters obtained from previous study, and the sensitivity of IgH rearrangements gene was examined by using SYBR green I real-time quantitative PCR. The results indicated that the optimal annealing temperature was 60 degrees C, the optimal concentration of primers was 0.8 micromol/L, the satisfactory Taq enzyme amount was 0.5 U, the optimal concentration of dNTP was 100 micromol/L, the optimal concentration of Mg(2+) was 3.0 mmol/L, the suitable number of cycle was 40 cycles. Amplification of IgH rearrangement gene and detection of desired gene fluorescence signal on SYBR green I real-time PCR were performed. The sensitivity of IgH gene using this quantitative PCR was 10(4)/ml. It is concluded that the optimal reaction parameters for amplification of clonal IgH rearrangements gene by using PCR technique was determined, and stable and specific amplification of desired gene with consensus primers was performed. Basically, IgH rearrangement gene was successfully detected by SYBR green I real-time PCR.
Gene Rearrangement, B-Lymphocyte, Heavy Chain ; genetics ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Organic Chemicals ; chemistry ; Polymerase Chain Reaction ; methods ; Reproducibility of Results

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

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

OBJECTIVETo develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology