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1.
Recombinant human Fab-H chain and L chain were separately expressed as inclusion body using Escherichia coli. After solubilization of Fab-H chain and L chain by the reduction and S-alkyldisulphidation in 8 M urea, about 100 mg of purified Fab-H chain and about 160 mg of L chain could be obtained from 1 l of each culture by ion-exchange chromatogram in the presence of 8 M urea. Combination of the lyophilized Fab-H chain and L chain could be efficiently folded to native human Fab by using the stepwise dialysis method and the human Fab was purified with cation-exchange chromatogram. In the folding procedure, it was found that cysteamine and cystamine with positive charge were effective to improve the folding yield of human Fab. Moreover, from comparison of folding yield in the presence of ten kinds of additives, it was suggested that taurine was effective to improve the folding of human Fab. Consequently, we could obtain about 60 mg of folded human Fab from 1 l of each culture under the optimum conditions. 相似文献
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3.
Human laminin M chain (merosin): complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues 总被引:18,自引:5,他引:13
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Reetta Vuolteenaho Maria Nissinen Kirsi Sairdo Mary Byers Roger Eddy Harri Hirvonen Thomas B. Shows Hannu Sariola Eva Engvall Karl Tryggvason 《The Journal of cell biology》1994,124(3):381-394
4.
Characterization and humanization of a monoclonal antibody that neutralizes human leukocyte interferon: a candidate therapeutic for IDDM and SLE 总被引:5,自引:0,他引:5
We have developed a panel of murine monoclonal antibodies that recognize human interferon alpha. One of these mononclonal antibodies binds and neutralizes, with high affinity, all of seven tested recombinant human interferon alphas. This mononclonal antibody also neutralizes the interferon activity present in two independent pools of interferon alphas prepared following stimulation of human peripheral blood leukocytes. The complementary determining regions from this murine mononclonal antibody were transferred to a human IgG2 heavy chain and to a human kappa1 light chain. In addition, six (heavy chain) and two (light chain) amino acids were transferred from the framework regions. This generated a humanized mononclonal antibody that retained the specificity of the mouse parent. The humanized anti-interferon alpha antibody is a candidate therapeutic for those diseases, such as insulin-dependent diabetes, systemic lupus erythematosis, psoriasis and Crohn's disease, which are all characterized by pathological expression of interferon alpha. 相似文献
5.
Human HB4C5 hybridoma cells produce a lung cancer-specific IgM human monoclonal antibody (mAb). HB4C5 human mAb cross-reacts
with Candida cytochrome c (Cyt c) and carboxypeptidase (Cpase). Concanavalin A (ConA)-resistant variants of HB4C5 cells loss
the original light chain followed by expression of various new light chains at a high incidence (light chain shifting) (Tachibana
et al., 1996). HTD8 cells, one of the ConA-resistant variant subclones of HB4C5 cells, undergo the active light chain shifting
and produce various sublines, each of which stably secretes new mAb consisting of a new light chain and a HB4C5 heavy chain.
The new mAb exhibits altered antigen binding ability from that of the original antibody. We could expect that HTD8 cells can
be used as ‘a light chain stem cell line’ to improve antigen binding ability and specificity of established human mAbs. A
BD9D12 IgG human mAb recognizes lung cancer cells and cross-reacts with cytokeratin 8. Introduction of the heavy chain gene
of BD9D12 mAb into HTD8 cells resulted in establishment of various sublines which secreted various kinds of hybrid antibodies
consisting of different light chains derived from HTD8 subclones which underwent light chain shifting and a common IgG heavy
chain derived from BD9D12. These hybrid antibodies exhibited different or improved reactivities to Cyt, Cpase, cytokeratin
8 and various cancer cells from those of parental mAb, demonstrating that light chain shifting can be applied to improve the
affinity and specificity of human mAb.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
6.
Isolation of a cDNA clone for the human laminin-B1 chain and its gene localization. 总被引:2,自引:0,他引:2
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M Jaye W S Modi G A Ricca R Mudd I M Chiu S J O''''Brien W N Drohan 《American journal of human genetics》1987,41(4):605-615
A cDNA clone encoding the B1 chain of human laminin has been isolated from a human endothelial cell cDNA library. With use of this probe and a panel of rodent/human somatic-cell hybrids and in situ hybridization, the gene for the human laminin-B1 chain has been localized to chromosome 7, band q31. 相似文献
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8.
J chain in Rana catesbeiana high molecular weight Ig 总被引:2,自引:0,他引:2
C A Mikoryak M N Margolies L A Steiner 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(12):4279-4285
A polypeptide homologous to human and mouse J chain has been identified in the high molecular weight (HMW) Ig of the bullfrog, Rana catesbeiana. In previous studies, we had detected a component that was similar in size to mammalian J chains and that, relative to L chains, migrated rapidly to the anode in alkaline-urea PAGE; however, its mobility was less than that of mammalian J chains. We now demonstrate that this component is covalently linked to the H chain of R. catesbeiana HMW Ig. All of the disulfide bridges of this polypeptide, like those of human and mouse J chain, can be cleaved by reducing agents even in the absence of denaturing solvents. The putative frog J chain was isolated by a procedure that did not require preliminary purification of the HMW Ig. The chain differed in amino acid composition from L chains but resembled J chains from several other species. Tryptic peptides were isolated and sequenced. Except for a single heptapeptide, the peptides could be aligned by virtue of their similarity to segments of human and mouse J chain. Of the 116 residues that were placed, 55 were identical with residues in human J chain and 60 with residues in mouse J chain. The six cysteine residues identified in the frog J chain are at the same positions as six of the eight cysteines in the human and mouse J chains. The results indicate significant conservation in structure between amphibian and mammalian Ig J chains. 相似文献
9.
Direct identification of class II histocompatibility DR proteins in preparations of human T-cell lymphotropic virus type III 总被引:9,自引:3,他引:6
L E Henderson R Sowder T D Copeland S Oroszlan L O Arthur W G Robey P J Fischinger 《Journal of virology》1987,61(2):629-632
Class II histocompatibility DR antigen alpha and beta chains were isolated from preparations of human T-cell lymphotropic virus type III grown in human H-9 cells. The proteins were purified by reversed-phase high-pressure liquid chromatography and identified by direct N-terminal amino acid sequence analysis of each chain. The purified DR alpha chain had an N-terminal amino acid sequence identical to the known sequence of human DR alpha chain through the first 37 residues. The N-terminal amino acid sequence of the purified DR beta chain was identical to that of human DR4 beta chain. The DR alpha and beta chains appeared to be identical to the p34-36K and p30-32K proteins, respectively, concentrated in immunostimulatory complexes prepared from unfractionated virus and were the major immunogens in these complexes. These proteins represent a ready source of antigens which can cause false-positive enzyme-linked immunosorbent assay reactions in individuals previously exposed to allogenic histocompatibility antigens. The removal of the DR chains from virus preparations by use of available monoclonal antibodies or other means should result in a lower rate of initial false-positive enzyme-linked immunosorbent assay reactions. 相似文献
10.
In this paper we present a procedure for the isolation of the native bovine alpha chain. The method is based on affinity chromatography. The results show that the ligand-binding properties of the bovine alpha chain are almost identical to those of the human alpha chain. The hybrid alphaB2 betaH2 prepared by mixing bovine alpha chains and human beta chains shows ligand binding properties similar to those of human hemoglobin and different from those of bovine hemoglobin. 相似文献
11.
Robert Wade David Feldman Peter Gunning Larry Kedes 《Molecular and cellular biochemistry》1989,87(2):119-136
12.
Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas. 相似文献
13.
Sequence homology and immunologic cross-reactivity of human cytomegalovirus with HLA-DR beta chain: a means for graft rejection and immunosuppression 总被引:15,自引:2,他引:13
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A peptide (Leu-Gly-Arg-Pro-Asp-Glu-Asp-Ser-Ser-Ser-Ser-Ser-Ser-Ser-Cys) that was identical to residues 82 through 96 of a predicted protein of 208 amino acids from the immediate-early region (IE-2) nucleic acid sequence of human cytomegalovirus was chemically synthesized. By computer analysis, the first five amino acids of this peptide showed sequence homology to the beta chain of the human histocompatibility complex HLA-DR. The homologous amino acids, 53 through 57, were located in a region that is conserved between the human DR beta chain and the beta chain of the H-2 class II histocompatibility antigen for mice. The shared region between the IE-2 protein and DR beta chain were similar in both hydrophilicity and predicted beta-turn potential. The IE-2 viral peptide induced antibodies that specifically recognized the human DR beta chain. These observations describe a protein encoded by the IE-2 region of human cytomegalovirus that contains sequence homology and shows immunologic cross-reactivity with a conserved domain of HLA-DR and suggest a mechanism to explain how human cytomegalovirus infection contributes to graft rejection after transplantation. 相似文献
14.
L. S. Young I. S. Bevan M. A. Johnson P. I. Blomfield T. Bromidge N. J. Maitland C. B. Woodman 《BMJ (Clinical research ed.)》1989,298(6665):14-18
The polymerase chain reaction is an in vitro method for primer directed enzymatic amplification of specific target DNA sequences. The technique was used to detect human papillomavirus types 11 and 16 simultaneously in cellular DNA recovered from cervical smears in 38 women referred for colposcopy to evaluate cytological abnormality and 10 women with no history of cytological abnormality. The polymerase chain reaction was shown to be both specific and sensitive in detecting human papillomavirus DNA such that a single human papillomavirus molecule was detected in 10(5) cells. Of the 38 women with cytological abnormality, all were positive for human papillomavirus on testing with the polymerase chain reaction; 36 were infected with human papillomavirus type 16 and 22 dually infected with human papillomavirus types 11 and 16. Seven of the 10 women with no cytological abnormality were also infected with human papillomavirus type 11 or 16. The use of the polymerase chain reaction will facilitate epidemiological investigation of the aetiological role of human papillomavirus in cervical neoplasia. This preliminary analysis suggests that the prevalence of human papillomavirus infection is greater than previously reported. 相似文献
15.
Globin prepared from hemoglobin of adult tupai (Tupaia glis) was separated into alpha and beta polypeptide chains by CM-cellulose column chromatography. The S-aminoethylated alpha polypeptide chain and S-carboxymethylated beta polypeptide chain were each digested with trypsin, and the sequences of all the peptides thus obtained were established. The ordering of these tryptic peptides in the alpha and beta polypeptide chains was deduced from the homology of their primary structures with that of human adult hemoglobin. In this way the primary structures of the alpha and beta polypeptide chains of tupai hemoglobin were established; 27 amino acids in the alpha polypeptide chain and 26 in the beta chain differ from those in human adult hemoglobin. 相似文献
16.
Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA. 总被引:4,自引:1,他引:3
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M Seno T Kurokawa Y Ono H Onda R Sasada K Igarashi M Kikuchi Y Sugino Y Nishida T Honjo 《Nucleic acids research》1983,11(3):719-726
DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2. 相似文献
17.
1. Human high molecular weight (HMW)-kininogen was highly purified from human plasma by chromatographies on QAE-Sephadex A-50 and CM-Sephadex C-50. Human HMW-kininogen thus purified was a mixture of a single chain and a disulfide-linked pair of chains. Human HMW-kininogen is an acidic glycoprotein having a molecular weight of 120,000. The amino acid composition of human HMW-kininogen is quite similar to that of bovine HMW-kininogen. 2. We investigated whether the liberation of kinin from human HMW-kininogen by human plasma kallikrein was accompanied by liberation of histidine-rich fragments, as observed with bovine HMW-kininogen (Han et al. (1975) J. Biochem. 77, 55--68). After prolonged incubation of human HMW-kininogen and human plasma kallikrein followed by gel-filtration on Sephadex G-50, a fragment of molecular weight 8,000 was isolated together with bradykinin. However, the histidine content of the fragment was not as high as that in the bovine fragments. Most of the histidine in human HMW-kininogen was recovered in the kinin-free protein, and the light chain of kinin-free protein was found to be rich in histidine compared with the heavy chain. These results suggest that the histidine-rich sequence in human HMW-kininogen is not released by the action of human plasma kallikrein, but remains bound to the light chain of kinin-free protein. 相似文献
18.
T Kurokawa M Seno R Sasada Y Ono H Onda K Igarashi M Kikuchi Y Sugino T Honjo 《Nucleic acids research》1983,11(10):3077-3085
Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed. These epsilon chain peptides were synthesized in E. coli under the control of the trp promoter-operator. The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids. 相似文献
19.
F D Finkelman J A van Boxel R Asofsky W E Paul 《Journal of immunology (Baltimore, Md. : 1950)》1976,116(4):1173-1181
A substantial fraction of human cord blood and peripheral blood lymphocytes have recently been shown to bear IgD. Although IgD has not been identified in mice, it has been suggested that it is also a major surface immunoglobulin of murine lymphocytes. Thus, lactoperoxidase-catalyzed iodination of surface immunoglobulin of mouse spleen and lymph node cells reveals the existence of an IgH chain differing from mu, gamma, and alpha-chain both antigenically and by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This new H chain class has been previously proposed to be the mouse homologue of delta-chain. In this paper, we analyzed human, mouse, guinea pig, and rabbit lymphoid cell membrane Ig by lactoperoxidase-catalyzed iodination, extraction with non-ionic detergent precipitation with a variety of specific anti-Ig sera, and electrophoresis of dissolved reduced precipitates on sodium dodecyl sulfate-polyacrylamide gels. Our studies confirm the previous reports of a new mouse cell membrane H chain with a mobility more rapid than that of mu-chain. However, we fail to detect a molecule with this electrophoretic mobility on the surface of guinea pig or rabbit lymph node and spleen cells. Moreover, neither anti-kappa nor anti-delta antibody precipitates a molecule with an H chain of this mobility from labeled extracts of human cord blood or peripheral blood lymphocytes. Cell surface delta was identified on both human cord blood and peripheral blood lymphocytes, but it proved to have mobility similar to human and mouse mu-chain. This result indicates either that mouse delta-chain has an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels which differs appreciably from that of human membrane delta-chain or that the newly described mouse H chain is not the homologue of human delta-chain. 相似文献
20.
We examined the extranuclear effects of thyroid hormones on human platelets. Pretreatment with DL-thyroxine or DL-triiodothyronine inhibited collagen-induced aggregation, in a dose-dependent manner, but other derivatives of thyroid hormone had no significant effects. In contrast to collagen, 12-O-tetradecanoylphorbol-13-acetate-induced aggregation was not affected by thyroid hormones at the same concentration range. Thyroxine also inhibited the release of [14C] serotonin from collagen-stimulated platelets, with a marked reduction in the phosphorylation of 20,000-dalton protein. Thyroxine and triiodothyronine had inhibitory effects on myosin light chain kinase purified from human platelets and inhibited more markedly the myosin light chain kinase than protein kinase C (Ca2+/phospholipid-dependent enzyme) and cAMP-dependent protein kinase. In addition, L-thyroxine behaved as a competitive inhibitor of myosin light chain kinase toward calmodulin, and the Ki value was calculated to be 2.6 microM. To determine whether or not thyroxine directly binds myosin light chain kinase, we prepared an affinity column, using L-thyroxine as the ligand. Myosin light chain kinase was selectively bound to the column while calmodulin passed through. We also designed a procedure for the purification of myosin light chain kinase from human platelets, using L-thyroxine-affinity chromatography. A markedly increased purification was thus achieved, and DEAE-cellulose and L-thyroxine-affinity chromatography were made feasible. These results suggest that thyroxine can serve as a pharmacological tool for elucidating the biological significance of myosin light chain kinase-mediated reactions and is a pertinent ligand which can be used to purify myosin light chain kinase from platelets as a substitute for calmodulin. 相似文献