AIM: To obtain evidence for selection of antigens used in genetically engineered vaccine against Helicobacter pylori (H pylori). METHODS: Enzyme linked immunoabsorbent assay (ELISA) was established on the basis of rec...AIM: To obtain evidence for selection of antigens used in genetically engineered vaccine against Helicobacter pylori (H pylori). METHODS: Enzyme linked immunoabsorbent assay (ELISA) was established on the basis of recombinant protein antigens rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB of H pylori to detect expression rates of the antigens in bacterial isolates as well as positive rates of the antibodies in sera from H pylori-infected patients. PCR was applied to the detection of carrying rates of the genes encoding antigens in the isolates. RESULTS: The outputs of rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB were approximately 35%, 32%, 15%, 23%, 56%, 25% and 20% of the total bacterial proteins, respectively. One hundred and fifty-one strains of H pylori were isolated from 347 biopsy specimens of chronic gastritis, peptic ulcer or gastric adenocarcinoma, with a positive rate of 43.5%. All of the isolates expressed UreB, HpaA, FlaA and FlaB while 52.3%, 92.1% and 93.4% of the isolates expressed VacA, CagA and NapA, respectively. In the sera of 151 H pylori-infected patients, the positive ratesof IgG antibodies against UreB, HpaA, VacA, CagA, NapA, FlaA and FlaB were 100%, 87.4%, 43%, 71.5%, 89.4%, 84.8% and 79.5%, respectively. Furthermore, the expression frequencies of VacA and NapA were found to be relative to the severity of gastric diseases (P = 0.016 and P < 0.0001, respectively). CONCLUSION: UreB antigen is the top option of developing genetically engineered vaccine against H pylori followed by NapA or HpaA.展开更多
AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search f...AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.展开更多
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
Recently we constructed a murine IgE phage surfacedisplay library and screened out two IgE (Fab) cloneswith specific binding activity to Trichosanthin (TCS). Inthis work, the Vε and Vκ genes of the two clones werese...Recently we constructed a murine IgE phage surfacedisplay library and screened out two IgE (Fab) cloneswith specific binding activity to Trichosanthin (TCS). Inthis work, the Vε and Vκ genes of the two clones weresequenced and their putative germline gene usages werestudied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carriedout to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCSrecognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments werealso analyzed respectively.展开更多
In the present study, 27 multi-drug resistant strains of Pseudomonas aeruginosa were isolated from clinical specimens in our hospital from Jan 2005 to Nov 2005, in which the resistant genes encoding β-lactamase inclu...In the present study, 27 multi-drug resistant strains of Pseudomonas aeruginosa were isolated from clinical specimens in our hospital from Jan 2005 to Nov 2005, in which the resistant genes encoding β-lactamase including TEM, SHV, OXA, PER, VEB, GES, CARB, IMP, VIM, SPM, GIM, DHA and OprD2 were tested by PCR amplification and sequenced by DNA sequencer. It was found that the detection rates of blaVEB, blaGES and blaCARB genes in these 27 isolates of P. aeruginosa were 11.1%, 11.1% and 48.1%, respectively, but almost the oprD2 gene was lacked （92.6%）. In addition, the resistant genes encoding TEM, SHV, OXA, PER, IMP, VIM, SPM, GIM and DHA β-lactamase were all not found. It was also demonstrated that the sequence of blaVEB gene appeared to be identical to that of the blaVEB-1 （AY536743）, while the blaGES and blaCARB genes shared 99% identity with blaGES-1 （AY219651） and blaCARB-3 （S46063） genes. From these observations, it is evident that P. aeruginosa carrying the blarEs, blaGES and blaCARB resistant genes isolated in our hospital confers the resistance to β- lactams, and the loss of the oprD2 gene may be the important cause to develop resistance to imipenem in P. aeruginosa.展开更多
Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study ...Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study of the allergic-specifc antibody responses. Here we report the construction of a normal human IgE combinatorial library The repertoire of IgE VH genes and of K genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR, and were then constructed to form the phage surface display human Fab(IgEVH) library. A plant protein allergen, trichosanthin(TCS), was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library. Human IgE(Fab) to TCS were detected.展开更多
In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating...In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.展开更多
基金The Natural Science Foundation of Zhejiang Province, No. Y207696
文摘AIM: To obtain evidence for selection of antigens used in genetically engineered vaccine against Helicobacter pylori (H pylori). METHODS: Enzyme linked immunoabsorbent assay (ELISA) was established on the basis of recombinant protein antigens rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB of H pylori to detect expression rates of the antigens in bacterial isolates as well as positive rates of the antibodies in sera from H pylori-infected patients. PCR was applied to the detection of carrying rates of the genes encoding antigens in the isolates. RESULTS: The outputs of rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB were approximately 35%, 32%, 15%, 23%, 56%, 25% and 20% of the total bacterial proteins, respectively. One hundred and fifty-one strains of H pylori were isolated from 347 biopsy specimens of chronic gastritis, peptic ulcer or gastric adenocarcinoma, with a positive rate of 43.5%. All of the isolates expressed UreB, HpaA, FlaA and FlaB while 52.3%, 92.1% and 93.4% of the isolates expressed VacA, CagA and NapA, respectively. In the sera of 151 H pylori-infected patients, the positive ratesof IgG antibodies against UreB, HpaA, VacA, CagA, NapA, FlaA and FlaB were 100%, 87.4%, 43%, 71.5%, 89.4%, 84.8% and 79.5%, respectively. Furthermore, the expression frequencies of VacA and NapA were found to be relative to the severity of gastric diseases (P = 0.016 and P < 0.0001, respectively). CONCLUSION: UreB antigen is the top option of developing genetically engineered vaccine against H pylori followed by NapA or HpaA.
基金Supported by the Major State Basic Research Development Program of China, 973 Program, No. 2002CB513100
文摘AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.
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.
文摘Recently we constructed a murine IgE phage surfacedisplay library and screened out two IgE (Fab) cloneswith specific binding activity to Trichosanthin (TCS). Inthis work, the Vε and Vκ genes of the two clones weresequenced and their putative germline gene usages werestudied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carriedout to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCSrecognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments werealso analyzed respectively.
文摘In the present study, 27 multi-drug resistant strains of Pseudomonas aeruginosa were isolated from clinical specimens in our hospital from Jan 2005 to Nov 2005, in which the resistant genes encoding β-lactamase including TEM, SHV, OXA, PER, VEB, GES, CARB, IMP, VIM, SPM, GIM, DHA and OprD2 were tested by PCR amplification and sequenced by DNA sequencer. It was found that the detection rates of blaVEB, blaGES and blaCARB genes in these 27 isolates of P. aeruginosa were 11.1%, 11.1% and 48.1%, respectively, but almost the oprD2 gene was lacked （92.6%）. In addition, the resistant genes encoding TEM, SHV, OXA, PER, IMP, VIM, SPM, GIM and DHA β-lactamase were all not found. It was also demonstrated that the sequence of blaVEB gene appeared to be identical to that of the blaVEB-1 （AY536743）, while the blaGES and blaCARB genes shared 99% identity with blaGES-1 （AY219651） and blaCARB-3 （S46063） genes. From these observations, it is evident that P. aeruginosa carrying the blarEs, blaGES and blaCARB resistant genes isolated in our hospital confers the resistance to β- lactams, and the loss of the oprD2 gene may be the important cause to develop resistance to imipenem in P. aeruginosa.
文摘Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study of the allergic-specifc antibody responses. Here we report the construction of a normal human IgE combinatorial library The repertoire of IgE VH genes and of K genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR, and were then constructed to form the phage surface display human Fab(IgEVH) library. A plant protein allergen, trichosanthin(TCS), was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library. Human IgE(Fab) to TCS were detected.
文摘In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.
