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Objective To develop a quantitative immunohistochemistry assay for duck hepatitis B virus core antigen (DHB-cAg)in duck liver tissue.Methods By comparison with no repair antigen and repair antigen with high pressure,microwave and trypsin,the best solution of antigen retrieval was determined.By optimizing the parameter of image acquisition and de-ducting blank area,mean density of yellow areas was calculated using Image-Pro Plus 6.0 software.Using the assay devel-oped to determine the level of DHBcAg in liver tissue from duck infected by DHBV,anti-DHBV activity of DHBcMAb-TAT PTD conj ugate was examined.Results SABC method with no repair antigen was selected,which was better than other methods.DHBcAg expression in duck liver tissue could be objectively and accurately quantified by setting Image-Pro Plus 6.0 software parameters and calculating mean density of yellow areas.By comparison with the differences between mean densityat baseline of treatment and end of treatment,it was showed that DHBcMAb-TATPTD conjugate treatment dose-de-pendently reduced the levels of DHBcAg in liver tissue,which show that the assay developed could effectively evaluate the anti-DHBV activity of agent.Conclusion The immunohistochemistry assay developed in this study can objectively and accu-rately evaluate the level of DHBcAg in duck liver tissue.
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OBJECTIVE@#To construct a recombinant prokaryoticexpression plasmid pET/ c-Aβ(15)-c, and evaluate the immunogenicity of its encoded fusion protein as expressed in E.coli.@*METHODS@#The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The synthetic, double-strand Aβ(1-15) gene was inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-71- Aβ(15) was spliced to HBc88-144, yielding the recombinant gene c-Aβ(15)-c; that gene was subcloned into pET-28a(+). The fusion protein (CA15C) expressed in the transformed E.coli BL21 was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The virus-like particle (VLP) formed by fusion protein CA15C was observed with transmission electric microscope (TEM). Four Kunming (KM) mice were given intraperitoneal injections of CA15C, and the anti-Aβ antibody elicited was detected by indirect ELISA.@*RESULTS@#The sequence of the recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, the fusion protein was expressed, mainly in the sediment from the bacterial lysate. The expression level was 40% of total protein in the sediment. The CA15C could form VLP. After 5 rounds of immunization, the titer of anti-Aβ antibody in the sera of KM mice reached 1:10000, while the anti-HBc antibody was undetectable.@*CONCLUSION@#Recombinant c-Aβ(15)-c gene can be expressed in E.coli. The expressed protein can form VLPs and has a strong immunogenicity.
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Animais , Humanos , Camundongos , Doença de Alzheimer , Peptídeos beta-Amiloides , Genética , Sequência de Bases , Vetores Genéticos , Genética , Antígenos do Núcleo do Vírus da Hepatite B , Genética , Alergia e Imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos , Genética , Proteínas Recombinantes de Fusão , Genética , Alergia e Imunologia , Metabolismo , Vacinas de Partículas Semelhantes a Vírus , Genética , Alergia e Imunologia , MetabolismoRESUMO
ObjectiveLoss-of-function mutation of p53,a tumor suppressor gene,is an important mechanism for the development of human cancers.In this study we tried to transfect p53N15-based fusion peptide into H1299,a lung cancer cell line,and evaluate the anti-tumor effects of the fusion peptide.MethodsAdeno-associated virus ( AAV) vectors were used for transfecting p53N15 fusion peptide into p53-null lung adenocarcinoma H1299 cells.The anti-replication effects of p53N15 fusion peptide were evaluated with inverted microscopy,MTT test for cell viability and flow cytometry.ResultsFusion peptides in H1299 cells was highly expressed and had detectable suppressive effects on cell proliferation.A large amount of dead cells were seen under microscope after the transfection of recombinant viruses for 72 hours.Cells activity was reduced significantly in the virus-transfecting groups as demonstrated by MTT test.The flow cytometry showed that a large number of dead cells were present in the virus-transfecting groups.ConclusionThe growth of H1299 lung adenocarcinoma cells could be inhibited in vitro by being transfected with p53N15 fusion peptide,which may be a potential gene therapy alone or as an adjuvant option in the treatment of lung cancer.
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OBJECTIVE@#To construct an universal recombinate adeno-associated virus (AAV), rAAV-NT4-ADNF-9, and to detect the ability of transfection of the rAAV vector into cochlea in vitro.@*METHOD@#pSSVHG-CMV-ADNF-9 plasmid was introduced into 293 cell by method of Ca3 (PO4)2 using three plasmids of pSSHG-CMV-NT4-ADNF-9, pFG140 and pAAV/Ad. The recombinate adeno-associated virus (rAAV) was harvested, and the titrations of the rAAV concentrated was detected by dot-blot test. Isolate and culture the cochlear hair cell of SD rats newly born in vitro. The rAAV-NT4-ADNF-9 was added to the medium while plating. Cochlear were collected 24 h after cultivation for RT-PCR to detect the transfection of rAAV-NT4-ADNF-9.@*RESULT@#The titration of rAAV stock produced 2 x 10(16) total particles/L, which showed that rAAV-NT4-ADNF-9 was constructed successfully. The cochlear hair cell of SD rats newly born was isolated and cultured in vitro successfully. It certified that rAAV-NT4-ADNF-9 was able to transfect into cochlear and express secretory NT4-ADNF-9 peptide by RT-PCR.@*CONCLUSION@#The rAAV vector constructed in this paper, rAAV-NT4-ADNF-9, can transfer into cochlear cultured in vitro, which layed a foundation of further research for gene therapy.
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Animais , Ratos , Células Cultivadas , Cóclea , Biologia Celular , Dependovirus , Genética , Vetores Genéticos , Células Ciliadas Auditivas , Biologia Celular , Proteínas do Tecido Nervoso , Genética , Ratos Sprague-Dawley , TransfecçãoRESUMO
Objective It was reported that p53 apoptotic peptide (N37) could inhibit p73 gene through being bound with iASPP, which could induce tumor cell apoptosis. To further explore the function of N37, we constructed the cloning plasmid of DNA fragment encoding p53 (N37) apoptotic peptide by using DNA synthesis and molecular biology methods. Methods According to human p53 sequence from the GenBank database, the primer of p53(N37) gene was designed using Primer V7.0 software. The DNA fragment encoding p53 (N37) apoptotic peptide was amplified by using self-complementation polymerase chain reaction (PCR) method and cloned into the pGEM-T Easy vector. The constructed plasmid was confirmed by endonuclease analysis and sequencing. Results The insertion of objective DNA fragment was confirmed by plasmid DNA enzyme spectrum analysis, p53 (N37) gene was successfully synthesized chemically in vitro. The sequencing result of positive clone was completely identical to the human p53(N37) sequence in GenBank using BLAST software (http://www. ncbi. him. nih. gov/cgi-bin /BLASTn). Conclusion The cloning of DNA fragment encoding p53(N37) apoptotic peptide was constructed by using DNA synthesis and pGEM-T Easy cloning methods. With the constructed plasmid, we could further investigate the function of N37 peptide.
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Objective To construct NT4-GFP-Ant fusional reporting gene and the vector of NT4-GFP-Ant recombinant adeno-associated virus(AAV).Methods The GFP gene was cloned by using PCR and T-vector cloning method.The positive clone was identified by the restriction enzymes,and then the cloned amplified fragments were sequenced and analyzed.The resulting gene of GFP and Ant,PBV220/NT4 were connected by DNA ligase,and thus PBV220/NT4-GFP-Ant was constructed,then the NT4-GFP-Ant fragment was gained and identified by the restriction enzymes.The resulting gene of NT4-GFP-Ant fragment was inserted into the EcoRⅠ-BamHⅠsite of vector plasmid pSSHG to construct the vector of NT4-GFP-Ant recombinant AAV.Results A 730 bp fragment of DNA was gained when T-easy/GFP was cut by the restriction enzyme EcoRⅠ.The cloned GFP gene was coincident with the sequence in GenBank.A 1 000 bp fragment of DNA was gained when pBV220/NT4-GFP-Ant was cut by the restriction enzymes BamHⅠ/EcoRⅠ.A 1 000 bp fragment of DNA was gained when PSSHG/NT4-GFP-Ant was cut by the restriction enzymes BamHⅠ/EcoRⅠ.Conclusion GFP gene is cloned successfully,NT4-GFP-Ant gene and PSSHG/NT4-GFP-Ant recombinant AAV vector are constructed successfully.
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Brassinosteroid (BR) and gibberellin (GA) are two groups of plant growth regulators essential for normal plant growth and development. To gain insight into the molecular mechanism by which BR and GA regulate the growth and development of plants, especially the monocot plant rice, it is necessary to identify and analyze more genes and proteins that are regulated by them. With the availability of draft sequences of two major types, japonica and indica rice, it has become possible to analyze expression changes of genes and proteins at genome scale. In this review, we summarize rice functional genomic research by using microarray and proteomic approaches and our recent research results focusing on the comparison of cDNA microarray and proteomic analyses of BR- and GA-regulated gene and protein expression in rice. We believe our findings have important implications for understanding the mechanism by which BR and GA regulate the growth and development of rice.
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Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Fisiologia , Giberelinas , Metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oryza , Genética , Fisiologia , ProteômicaRESUMO
Objective To prepare a hybridoma secreting stab le monoclonal antibodies against ?-amyloid peptide (A? 1-42) with high titer. Methods By genetic engineering technology, A ? gene was recombined with the MIR of HBcAg to get the A? and HBcAg fusi on protein. Spleen cells from BALB/c mice immunized with A? and HBcAg f usion protein were fused with mouse myeloma cells SP2/0. Results Two strains of hybridomas (1H 7 and 1F 3) secreting stable monoclonal antibodies raised against A? 1-42 were ob tained. The subtypes of A? 1-42 antibodies were IgG 3. C onclusion The A? 1-42 monoclonal antibodies obtained have high titers and specificity.
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Objective To construct and expr es s a recombinant plasmid of nonstructural protein NS3-NS4 of hepatitis C virus ( HCV), and to identify the antigenicity of the expressed protein. Methods A gene region encompassing the nonstructural protei n NS3-NS4 of HCV was amplified by polymerase chain reaction (PCR) from the pUC1 9/HCV template. The recombinant expression plasmid containing the pBV220/NS3-NS 4 sequence was constructed, and the nonfused NS3-NS4 recombinant protein was ex pressed in E.coli DH5? efficiently. The recombinant protein was det ected by SDS-PAGE and ELISA. Results We successfully constructed and expressed the recom binant plasmid in prokaryote. Its antigenicity was detected with 50 standard ser a. Compared with the second-generation diagnostic Kit, the total detection rate was 96%. Conclusion The whole NS3-NS4 protein, a region of dominant immunogenicity, should be the effective component of the HCV diagnostic Kit and provide the clue for developing HCV DNA vaccine.
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Objective Cloning and sequencing of the human neurotrophin-4(hNT-4) gene.Methods With the chromosomal DNA of human blood lymphocytes as template,hNT-4 coding genes were amplified by polymerase chain reaction(PCR) and recombinated into phage vector pGEM-T Easy,which were sequenced by using Sanger's single stranded DNA terminal termination method.Results The sequence of the cloned gene is completely the same as that reported in the literature(GenBank data base,M86528).Conclusion This study successfully cloning and sequenced the gene of mhNT-4,and it would be convenient for us to study the expression of mhNT-4 in eukaryote,and to continue the research on the gene therapy of Alzheimer's disease intensively.This study indicate that the hNT-4 is conservative in different races and individuals.
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Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous system. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plasmids were encapsidated as recombinant virions. PC12 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus of AAV vectors by Southern blot and transcript situation of foreign genes by dot blot. Results The hybridization tests showed that AAV introduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PC12cells. Conclusion AAV vectors can serve as high efficiency vectors of target genes in neuronal PC12 cells.
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Objective Expressing the human matured brain-derived neurotrophic factor (mBDNF) gene in E.Coli and determining its bioactivity. Methods The resulting gene of mBDNF was subcloned into the EcoRI-BamHI site of the expression vector plasmid pBV220. The ligation products were used to transform the competent E. Coli DH5α. The proteins of mBDNF were experessed by temperature inducing. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the embryonic chicken DRG to test whether the expressed mBDNF is a biologically active protein. Results The recombinant plasmid pBV/mBDNF was successfully constructed. By temperature inducing,under the control of the bacteriophage λ PL promoter, the experessed mBDNF protein was a 14Kd non-fusion protein,which existed in E. Coli as inclusion bodies. The size of expressed mBDNF is identical to the prediction. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of embryonic 8-day-old chicken DRG neurons as compared to control.Conclusion The mBDNF gene can be expressed bioactively in E. Coli.
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ObjectiveTo study the cloning and sequencing of mature fragment of human bone morphogenetic protein-4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT- PCR method, the cDNA coding for the mature fragment of BMP-4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy-mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT-PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154 (201): G→C, which had no influence on the corresponding amino acids (Val). Another was at basel222 (269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.
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ObjectiveTo express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of PRPL promoter of a high-level ex- pression vector pBV220. HCV core gene was expressed in E. coli in a non-fused form. The expression protein was analysed by SDS-PAGE , and its immunoactivity was tested by ELISA. Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS-PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14. 0% of the total bacterial proteins ap- peared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV pos- itive sera from patients with hepatitis C . Conclusion The intact HCV core protein was successfully expressed in E . coli in a non-fused form on a high level, and its immunoactivity was high.
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In the present study, we have cloned the gene of human neurotrophin-3 (hNT-3) from the genomic DNA of white blood cells (WBC) by polymerase chain reaction (PCR). The amplification products were cloned into pUC19 and sequenced. Genomic sequence comparison of the cloned fragment and the reported hNT-3 (GenBank M61180) reveals 7 base differences: 1 in the signal peptide, 3 in the prepro peptide, and 3 in the mature hNT-3. Except the 2 varied bases (16th, T to G; 285th, A to C) in the signal peptide and pro-sequence resulted in the change of their encoded amino-acids (Tyr→Asp; Gln→His), the other varied bases have no influence on their respective encoded amino-acids, and all the changes have no influence on the open reading frame (ORF) of the hNT-3.
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Objective:To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant,laying a foundation for gene therapy research of malignant tumors.Methods:The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme.The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus;the products were cotransfered into HEK-293 cell line with helper plasmid PJM17.The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming.The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction(RT-PCR)procedure.The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Results:The p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing.High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells(1?10 11pfu/ml).The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR.Ad.NT4p53Ant had strong killing effect on HepG2 cells.Compared with Ad.GFP,Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells.Conclusion:The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques,laying a foundation for further research of gene therapy of cancer.
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Objective: To study the effects of physical and chemical factors on hairy root of Panax ginseng and its glycoside production. Methods: The cell growth index and its glycoside yield in different culture condition such as different plant hormones mixed, mediums and time were compared. Results: After hairy root of Panax ginseng was precultivated on the MS medium with IBA (0.5%) at first 72 hours. the culture was cultured on the MS medium. During 6 weeks, dry weight of hairy root increased by 98.74 times, and the content of glycoside synthesized by hairy root is 5.190% and Re+Rg1 is 0.3271%, respectively. Conclusion:We have achieved the best condition of hairy root of Panax ginseng.
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Objective To construct the expression box of fusion gene NT4-p53(N15)-Ant.Methods The gene of p53(N15)-Ant was obtained by PCR of two primers templating with each other and T-vector cloning method.The positive clone was identified and analyzed by the restriction enzymes and sequencing respectively.After digested with restriction enzyme,the interest gene of p53(N15)-Ant was subcloned into the plasmid pBV220/NT4.Results The gene of p53(N15)-Ant was confirmed by the digestion of restriction enzyme and sequencing.The recombinant plasmid pBV220/NT4p53(N15)Ant was identified by the digestion of restriction enzyme and agarose gel electrophoresis and the results conformed theoretical values.Conclusion The plasmid pBV220 containing the expression box of NT4-p53(N15)-Ant was successfully constructed by molecular cloning and recombination techniques in vitro,which will guide further study on gene therapy of cancer.
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Objective To express the hNGF? gene segment encoding mature peptide (hNGF? ) in E.coli and determine its bioactivity. Methods The resulting gene of hNGF? was subclonedinto the hNGF? site of the expression vector plasmid pBV220. The ligation products were used to transform the competent E.coli DH 5?. The proteins of hNGF? were expressed by temperature induction. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the expressed hNGF? tests of neurite growth of dorsal root knot of chicken embryo and tests of Brdu incorporation into PC12 cells was a biologically active protein. Results The recombinant plasmid pBV220/NGF? was successfully constructed. The NGF? was inserted to pBV220 plasma, a prokaryotic expression vector. Expression of NGF? in E.coli was induced by raising temperature to 42℃. SDS-PAGE electrophoresis showed that NGF? protein existed in inclusion. The solubility protein of NGF? was obtained through purification of inclusion by centrifugation and technique of protein repatriation. Recombinant NGF? protein was purified by affinity chromatography of heparin SepharoseCL-6B. The purity of NGF? was higher than 90% and yield of NGF? was 1.8~2.0mg/L expressing bacteria. The bioactivity of NGF? expressing prokaryotic cell was 1?10 5BU/g according to rule concerning examination of biological products in China. Conclusion The hNGF?gene with bioactivity can be expressed in E.coli.
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Objective To investigate survivin as an anticancer therapeutic target by use of shepherdin [79-87],a novel peptide carrying the survivin sequence from Lys-79 through Leu-87,we constructed an recombinant vector containing fusion gene NT4-Ant-Shepherdin [79-87].Methods The gene of Ant-Shepherdin [79-87] was obtained by PCR and T-vector method.After cloned and digested with restricted enzyme,Ant-shepherdin [79-87] was inserted in PBV220NT4 vector.The recombinant vector was transformed into the competent cell,E.coli DH5?.The fusion gene of NT4-Ant-Shepherdin [79-87] was identified by agarose gel electrophoresis (AGE).Results DNA sequencing results verified that the sequence of Ant-Shepherdin [79-87] was consistent with what we had designed.After transformed E.coli DH5?,a fragment of 321 bp was confirmed.Conclusion The recombinant vector containing fusion gene NT4-Ant-Shepherdin [79-87] was successfully constructed in this experiment by molecular biology techniques,which provides the basis of further research of survivin for cancer gene therapy.