The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by t...The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.展开更多
G protein-coupled receptors(GPCRs)are pivotal in mediating diverse physiological and pathological processes,rendering them promising targets for drug discovery.GPCRs account for about 40%of FDA-approved drugs,represen...G protein-coupled receptors(GPCRs)are pivotal in mediating diverse physiological and pathological processes,rendering them promising targets for drug discovery.GPCRs account for about 40%of FDA-approved drugs,representing the most successful drug targets.However,only approximately 15%of the 800 human GPCRs are targeted by market drugs,leaving numerous opportunities for drug discovery among the remaining receptors.Cell expression systems play crucial roles in the GPCR drug discovery field,including novel target identification,structural and functional characterization,potential ligand screening,signal pathway elucidation,and drug safety evaluation.Here,we discuss the principles,applications,and limitations of widely used cell expression systems in GPCR-targeted drug discovery,GPCR function investigation,signal pathway characterization,and pharmacological property studies.We also propose three strategies for constructing genome-wide pan-GPCR cell libraries,which will provide a powerful platform for GPCR ligand screening,and facilitate the study of GPCR mechanisms and drug safety evaluation,ultimately accelerating the process of GPCR-targeted drug discovery.展开更多
Currently,display-based methods are well established and widely used in antibody engineering for affinity maturation and structural stability improvement.We obtained a novel anti-human programmed death 1(PD-1)antibody...Currently,display-based methods are well established and widely used in antibody engineering for affinity maturation and structural stability improvement.We obtained a novel anti-human programmed death 1(PD-1)antibody using computer-aided design and a mammalian cell display technology platform.We used computer-aided modeling and distance geometry methods to predict and assign the key residues that contributed to the binding of human PD-L1 to PD-1.Then,we analyzed the sequence of nivolumab(an anti-human PD-1 antibody,referred to as MIL75 in the article)to determine the template for antibody design and library construction.We identified a series of potential substitutions on the obtained template and constructed a virtual epitope-targeted antibody library based on the physicochemical properties and each possible location of the assigned key residues.The virtual antibody libraries were displayed on the surface of mammalian cells as the antigen-binding fragments of full-length immunoglobulin G.Then,we used flow cytometry and sequencing approaches to sort and screen the candidates.Finally,we obtained a novel anti-human PD-1 antibody named FV78.FV78 competitively recognized the PD-1 epitopes that interacted with MIL75 and possessed an affinity comparable to MIL75.Our results implied that FV78 possessed equivalent bioactivity in vitro and in vivo compared with MIL75,which highlighted the probability and prospect of FV78 becoming a new potential antibody therapy.展开更多
Double fertilization is one of the predominant features of sexual reproduction in flowering plants but, because of the physical inaccessibility of gametes, the essential molecular mechanisms in these processes are lar...Double fertilization is one of the predominant features of sexual reproduction in flowering plants but, because of the physical inaccessibility of gametes, the essential molecular mechanisms in these processes are largely unknown. Based on the techniques for isolating highly purified gametes from several species and well-developed methods for manipulating RNA from limited quantities of gametes, genome-wide investigations of gamete transcription profiles were recently conducted in flowering plants. In this review, we survey the accumulated knowledge on gamete collection and purification, cDNA library construction, and transcript profile analysis to assess our understanding of the molecular mechanisms of gamete specialization and fertilization.展开更多
Using subtractive cDNA cloning method, a series of cDNA fragments have been isolated. One of them has been selected, that expressed differentially in the mouse intermediatelate erythroblasts, as a probe for the screen...Using subtractive cDNA cloning method, a series of cDNA fragments have been isolated. One of them has been selected, that expressed differentially in the mouse intermediatelate erythroblasts, as a probe for the screening of mouse erythroid differentiation related gene from a cDNA library constructed from mouse mid-late erythroids. A positive cDNA clone has been obtained using phage in situ hybridization coupled with PCR technique. Sequencing of this cDNA by utilizing the Bluescript-SK( + ) phage vector system demonstrates that it has a full length of 505 bp with an open reading frame of 309 bp (from 53 to 359 bp) encoded a protein of 102 amino acids. There is a tailing signal AATAA at 462 bp and a 26-bp long poly A tail terminal at the 3′UTR. Searches of GenBank databases failed to find any homologous sequences (GenBank Accession Number: Banklt 180846 AF 060220) and it was proved to be a novel gene designated mouse erythroid differentiation-denucleation related gene (MEDDF). Northern blot analysis展开更多
Early etiological diagnosis is very important for the control of sudden viral infections,and requires antibodies with both high sensitivity and high specificity.Traditional antibody preparation methods have limitation...Early etiological diagnosis is very important for the control of sudden viral infections,and requires antibodies with both high sensitivity and high specificity.Traditional antibody preparation methods have limitations,such as a long and arduous cycle,complicated operation,and high expenses.A chicken lymphoma cell line,DT40,is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture.Here,the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro.Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase(AID)gene,AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected.In this study,we generated a novel AID-inducible DT40 cell line(DT40-H7),in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system,and an inducible AID gene,based on the Tet-Off expression system,was stably transfected.AID expression was controlled in DT40-H7 cells in a simple and efficient manner;gene conversion and point mutations were observed only when AID was expressed.Using the antibody library generated from this cell line,we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus.The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.展开更多
以酵母穿梭表达质粒pGADT72Ree为载体，采用CLONTECH SMART（switching mechanism at 5’end of RNA transeript）技术，在酵母细胞中构建鸡法氏囊B细胞的酵母双杂交cDNA文库。通过从分离的鸡法氏囊B细胞中提取总RNA，利用经修饰的CDS...以酵母穿梭表达质粒pGADT72Ree为载体，采用CLONTECH SMART（switching mechanism at 5’end of RNA transeript）技术，在酵母细胞中构建鸡法氏囊B细胞的酵母双杂交cDNA文库。通过从分离的鸡法氏囊B细胞中提取总RNA，利用经修饰的CDS引物合成cDNA第一链，在链的末端可自动延伸CCC，在反应体系中加入SMART Ⅲ^TM Oligo作为cDNA第二链合成的引物，进行长距离（LD）2PCR合成双链eDNA，后者经CLONTECHCHROMASPIN＋TE24000柱纯化后，与线性质粒pGADT72Ree共转化感受态酵母菌AH109，二者在酵母细胞中利用酵母细胞内具有较高的同源重组酶活性，进行同源重组成环行的有复制活性的文库质粒，在缺亮氨酸（LEU）培养板上筛选出所有克隆，即为鸡法氏囊B细胞的酵母双杂交cDNA文库。结果：共获得2．2×10^6转化子，文库扩增后，插入cDNA长度在0．5—3kb。研究结论表明可在酵母细胞中利用CLONTECH SMART技术成功构建鸡法氏囊B细胞的酵母双杂交eDNA文库。展开更多
基金This work was supported by grants from the National Natural Science Foundation of China(No.30400111)the Natural Science Foundation of Jiangsu Province(No.BK2004041).
文摘The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.
基金supported by introducing the talented person scientific research starts funds subsidization project of Chengdu University of Traditional Chinese Medicine(030040019,030040017,China).
文摘G protein-coupled receptors(GPCRs)are pivotal in mediating diverse physiological and pathological processes,rendering them promising targets for drug discovery.GPCRs account for about 40%of FDA-approved drugs,representing the most successful drug targets.However,only approximately 15%of the 800 human GPCRs are targeted by market drugs,leaving numerous opportunities for drug discovery among the remaining receptors.Cell expression systems play crucial roles in the GPCR drug discovery field,including novel target identification,structural and functional characterization,potential ligand screening,signal pathway elucidation,and drug safety evaluation.Here,we discuss the principles,applications,and limitations of widely used cell expression systems in GPCR-targeted drug discovery,GPCR function investigation,signal pathway characterization,and pharmacological property studies.We also propose three strategies for constructing genome-wide pan-GPCR cell libraries,which will provide a powerful platform for GPCR ligand screening,and facilitate the study of GPCR mechanisms and drug safety evaluation,ultimately accelerating the process of GPCR-targeted drug discovery.
基金The work was supported by the National Natural Sciences Foundation of China grant(No.81272528 and No.31370938)National High Technology Research and Development Program(863 Program,No.2012AA02A302)+1 种基金National Science and Technology Major Projects for'Major New Drugs Innovation and Development'(2014ZX09304311-001-002-004)the Beijing Natural Science Foundation(No.5152022).
文摘Currently,display-based methods are well established and widely used in antibody engineering for affinity maturation and structural stability improvement.We obtained a novel anti-human programmed death 1(PD-1)antibody using computer-aided design and a mammalian cell display technology platform.We used computer-aided modeling and distance geometry methods to predict and assign the key residues that contributed to the binding of human PD-L1 to PD-1.Then,we analyzed the sequence of nivolumab(an anti-human PD-1 antibody,referred to as MIL75 in the article)to determine the template for antibody design and library construction.We identified a series of potential substitutions on the obtained template and constructed a virtual epitope-targeted antibody library based on the physicochemical properties and each possible location of the assigned key residues.The virtual antibody libraries were displayed on the surface of mammalian cells as the antigen-binding fragments of full-length immunoglobulin G.Then,we used flow cytometry and sequencing approaches to sort and screen the candidates.Finally,we obtained a novel anti-human PD-1 antibody named FV78.FV78 competitively recognized the PD-1 epitopes that interacted with MIL75 and possessed an affinity comparable to MIL75.Our results implied that FV78 possessed equivalent bioactivity in vitro and in vivo compared with MIL75,which highlighted the probability and prospect of FV78 becoming a new potential antibody therapy.
基金supported by the National Basic Research Program of China (Grant No. 2007CB108700)the National Natural Science Foundation of China (Grant Nos. 30821064 and 30771131)
文摘Double fertilization is one of the predominant features of sexual reproduction in flowering plants but, because of the physical inaccessibility of gametes, the essential molecular mechanisms in these processes are largely unknown. Based on the techniques for isolating highly purified gametes from several species and well-developed methods for manipulating RNA from limited quantities of gametes, genome-wide investigations of gamete transcription profiles were recently conducted in flowering plants. In this review, we survey the accumulated knowledge on gamete collection and purification, cDNA library construction, and transcript profile analysis to assess our understanding of the molecular mechanisms of gamete specialization and fertilization.
文摘Using subtractive cDNA cloning method, a series of cDNA fragments have been isolated. One of them has been selected, that expressed differentially in the mouse intermediatelate erythroblasts, as a probe for the screening of mouse erythroid differentiation related gene from a cDNA library constructed from mouse mid-late erythroids. A positive cDNA clone has been obtained using phage in situ hybridization coupled with PCR technique. Sequencing of this cDNA by utilizing the Bluescript-SK( + ) phage vector system demonstrates that it has a full length of 505 bp with an open reading frame of 309 bp (from 53 to 359 bp) encoded a protein of 102 amino acids. There is a tailing signal AATAA at 462 bp and a 26-bp long poly A tail terminal at the 3′UTR. Searches of GenBank databases failed to find any homologous sequences (GenBank Accession Number: Banklt 180846 AF 060220) and it was proved to be a novel gene designated mouse erythroid differentiation-denucleation related gene (MEDDF). Northern blot analysis
基金sponsored by the Project of the National Defense Science and Technology Innovation Special Zone (to HH, 17-163-12-ZT-005-013-01)the CAMS Innovation Fund for Medical Sciences (to HH, CIFMS 2016-12 M-1-013 and to ZZ, CIFMS 2016-12 M-1-014 and 2016-12 M-3-020)+1 种基金the National Key Research and Development Program (to ZZ, 2016YFD0500300)the Fundamental Research Funds for the Central Universities (to HH, 2018PT31032)
文摘Early etiological diagnosis is very important for the control of sudden viral infections,and requires antibodies with both high sensitivity and high specificity.Traditional antibody preparation methods have limitations,such as a long and arduous cycle,complicated operation,and high expenses.A chicken lymphoma cell line,DT40,is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture.Here,the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro.Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase(AID)gene,AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected.In this study,we generated a novel AID-inducible DT40 cell line(DT40-H7),in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system,and an inducible AID gene,based on the Tet-Off expression system,was stably transfected.AID expression was controlled in DT40-H7 cells in a simple and efficient manner;gene conversion and point mutations were observed only when AID was expressed.Using the antibody library generated from this cell line,we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus.The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.
