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Objective To construct vector expressing soluble mPDL1-hIgGFc and study its effect on the proliferation and apeptosis of cells in vitro. Methods The extrncellular domain of mPDL1 gene was amplified from pmPDL1 vector by PCR and inserted into phIgGFc vector. The recombinant pmPDL1-hIgGFc was transfected into CHO cells by LipofectAMINETM2000, and the transfected cells were named as CHOp. The expression of mPDL1-hIgGFc in the culture supernatants of CHOp was assayed by ELISA and Western blot. The effects of CHOp culture supernatants on mixed lymphocyte culture(MLC) was analysed by Flowm-etry. Results The extracellular domain of mPDL1 gene were obtained from PCR. DNA sequencing and the identification of digestion by HindⅢ and KpnⅠ indicated the recombinant plasmid pmPDL1-hIgGFc was suc-cessfully constructed. ELISA and Western blot analysis proved that the CHOp could express mPDL1-hIgG-Fc. CHOp culture supernatants could inhibit lymphocyte proliferation and induce the apoptosis of the activa-ted T cells in MLC in vitro in a dose-dependent manner. Conclusion The mPDL1-hIgGFc protein could in-hibit lymphocyte proliferation and induce the apoptosis of the activated T cells.
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The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDSPAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD=0.9921 ku),which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of IdHSP complex vaccine for B-CLL.
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Objective To explore the effects of SLC concentration gradient on suppression of tumor immune escape. Methods According to different SLC concentration, there were six groups. The HLA-Ⅰexpression and apoptosis of MCF-7 were detected with FCM,and intracellular BCL-2 expression was analyzed by western blot. The production of TGF-? was detected with ELISA. Results In a certain range of concentration gradient, following SLC increase, HLA-Ⅰexpression level on MCF-7 was improved, and apoptosis was induced but BCL-2 expression was enhanced. Moreover, the secretion of TGF-? was suppressed. Conclusion SLC inhibites tumor immune escape.
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The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.
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<p><b>OBJECTIVE</b>Sequences analysis of Ig variable regions from the peripheral blood mononuclear cells (PBMC) of patients with chronic B lymphocytic leukemia.</p><p><b>METHODS</b>Total RNA was isolated from PBMC of patients with chronic B lymphocytic leukemia, oligo-dT-primed cDNA was synthesized from RNA. The cDNA was amplified by Taq DNA polymerase with a set of specific 5' primers corresponding to Ig FR1 and 3' primers corresponding to CH1 (C micro /C) or CL (Ckappa/Clambda), the PCR products of variable regions of Ig heavy (IgH) and light (IgL) chains were sequenced by ABI PRISM Dye terminator cycle sequencing ready reaction kit and ABI PRISM 310 Genetic Analyzer. The gene homology of variable regions of IgH and IgL chains was compared by using DNA tools 5.1 system and "the international immunogenetics database".</p><p><b>RESULTS</b>Four light chains and 3 heavy chains were amplified from 4 and 3 patients respectively. Homology analysis of the sequences of 4 light chains and 3 heavy chains were performed by DNA tools system. The sequences of light chains are high homologous. And the sequences of heavy chains are quite different. The homologous analysis of the sequences of variable region by using "the international immunogenetics database" showed that the sequences were higher homologous to idiotype gene of some B lymphocytic leukemia. Four VL genes belong to human Ig Vkappa subgroup I, 2 of 3 VH genes belong to VH3 family and 1 belongs to VH5 family.</p><p><b>CONCLUSION</b>Ig genes have idiotype and same disease may have same idiotype.</p>
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Humanos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Química , Rearranjo Gênico , Genes de Imunoglobulinas , Fragmentos Fab das Imunoglobulinas , Genética , Cadeias Pesadas de Imunoglobulinas , Genética , Cadeias Leves de Imunoglobulina , Genética , Região Variável de Imunoglobulina , Genética , Leucemia Linfocítica Crônica de Células B , Genética , Alergia e Imunologia , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
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.
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Humanos , Sequência de Aminoácidos , Rearranjo Gênico , Região Variável de Imunoglobulina , Genética , Leucemia Linfocítica Crônica de Células B , Genética , Dados de Sequência Molecular , MutaçãoRESUMO
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.
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Sequência de Aminoácidos , Rearranjo Gênico , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Dados de Sequência Molecular , MutaçãoRESUMO
Objective:To study the effect of resistance to CTL mediated cytotoxity of target cells transfected with Wee1 and treated with anti perforin antibody Methods:Cytotoxic T lymphocyte was obtained by mixing lymphocyte NIT culture in vitro,and proliferation index (PI) of lymphocytes was detected(MTT method) The recombinant vector pCMV Wee1Hu/GFP was transfected into mammalian cells NIT With the function of anti perforin antibody, NIT and NIT transfected with Wee1 used as target cells, CTL mediated cytotoxity was detected by LDH method Results:CTL proliferation was induced by mixing cell culture With anti perforin antibody, the cytolysis quantity of NIT transfected with Wee1 was least Conclusion:Target cells transfected with Wee1 could resist CTL mediated cytotoxity, and anti perforin antibody obviously increased the effect
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Objective:To investigate the influence of dendritic cells modified by hIL-10 on proliferation and cytotoxicity of lymphocyte in experimental animals.Methods:The modified dendritic cells were injected intraperitoneally into C57BL/6 mice to sensitize it. Peripheral blood mononuclear cells (PBMC) of sensitized or unsensitized C57BL/6 mice act as reactive cells, dendritic cells modified or unmodified by hIL-10 act as stimulation cells. These two type cells co-cultured for 6 days. MTT assay was used to detect lymphocyte proliferation. Lactate dehydrogenase release method was used to examine the cytotoxic activity.Results:The results showed that IL-10 can inhibit lymphocyte proliferation reaction of unsensitized or sensitized C57BL/6 mice. Lymphocyte proliferation induced by modified dendritic cells is significant lower than that by unmodified dendritic cells. Modified dendritic cells can resist the cytotoxic activity of cytotoxic T lymphocyte.Conclusion:Dendritic cells stably expressing IL-10 can obviously decrease lymphocyte proliferation response of allogenic mice and reduce the cytotoxic activity.
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A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.
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A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.
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Objective To establish a rapid,sensitive and specific diagnostic test for detecting Neisse-ria gonorrhoea.Methods The major outer membrane proteins(P Ⅰ )in different gonococcal serogroups were obtained by isolation of outer membrane complex with CTB-ethanol precipitation,the outer membrane proteins were extracted with Z 3,14 and EDTA,and purified with DEAE-Sepharose CL-6B to obtain P Ⅰ .Hybridoma cell lines producing McAbs against P Ⅰ were established with lymphocyte hybridoma techniques.Results The molecular weight of P Ⅰ A and P Ⅰ B were determined with SDS-PAGE as35.2kDa and36.7kDa,respectively.Five hybridoma cell lines producing McAbs continuouslly and stably against P Ⅰ A and P Ⅰ B were obtained,in-cluding two hybridoma cell lines producing McAbs against P Ⅰ A and three hybridoma cell lines producing McAbs against P Ⅰ B.The titers of McAbs in the supernatants in the cultures and in abdominal ascites of BALB/c were from1:64to1:256and from1:4096to1:16384,respectively;and the specificity of the McAbs against P Ⅰ A and P Ⅰ B was so high that they easily reacted with N.gonorrhoeae but did not with other antigens such as N.meningitidis etc.Conclusion The purified P Ⅰ and the McAbs obtained in the study provide a basis to establish a rapid,sensitive and specific diagnostic test for detecting N.gonorrhoea.