RESUMO
Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vector. The recombinant plasmid pCJE-ME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJE-CME, the cell lines continuously expressing structural proteins could be selected with G418. And the expression products of pCJE-CME vector could be detected by ELISA, Western Blot and IFA assay. It showed that the JEV capsid protein could enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.
Assuntos
Animais , Cricetinae , Apoptose , Células CHO , Proteínas do Capsídeo , Genética , Metabolismo , Linhagem Celular , Biologia Celular , Virologia , Cricetulus , Vírus da Encefalite Japonesa (Espécie) , Genética , Metabolismo , Expressão Gênica , Vetores Genéticos , Proteínas Estruturais Virais , Genética , MetabolismoRESUMO
Based on the infectious clone of JEV vaccine SA14-14-2, the subgenomic replicons pCTCJEV, pCTMJEV with large deletions in the structural region were constructed. Then they were transfected into BHK-21 cell, the RNA replication of JEV subgenome can be monitored by RT-PCR and the non-structural protein can be found expressed in the cell by IFA. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive-strand and the negative-strand JEV RNA, we inserted an enhanced green fluorescence protein (EGFP) gene into the 3'-UTR of pCTCJEV, pCTMJEV under the control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA. After transfection, the EGFP fluorescence could be seen under the fluorescence microscope 1 day later,and maintained for more than a week with no apparent cytopathic effect. The constructed JEV replicons would provide valuable tools to provide a possible vector for a long-lasting RNA virus expression system.
Assuntos
Animais , Linhagem Celular , Vírus da Encefalite Japonesa (Espécie) , Genética , Vetores Genéticos , Genética , Proteínas de Fluorescência Verde , Genética , Metabolismo , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Genética , RNA Viral , Genética , Metabolismo , Replicon , Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Stable transformants for mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a newly screening vector plasmid pMCEscan, which carrying a FRT-neo*-IRES-k2tPA fusion gene and a DHFR gene, we screened colonies by k2tPA expression level. We selected 7 clones that expressed high level of k2tPA and carried one copy of the plasmid in their chromosomes. These clones showed in high level k2tPA production without amplification. So we targeted reporter gene (k2tPA) to test the basal expression ability of these cells clones. The clone, 8-1, showed the same effect to high base expression level. In this clone, the FRT-neo*-IRES-tPA gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a FRT-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal FRT by FLP recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. Using the site-specific integration CHO/dhfr- cell line 8-1, the expression level of k2tPA could amount to 17.1 microg/10(6) cell x 24 h.
Assuntos
Animais , Cricetinae , Células CHO , Cricetulus , DNA Nucleotidiltransferases , Genética , Amplificação de Genes , Marcação de Genes , Métodos , Genes Reporter , Engenharia Genética , Métodos , Proteínas Recombinantes , Genética , Tetra-Hidrofolato Desidrogenase , GenéticaRESUMO
A completely synthetic gene encoding the He domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, -50 kD) was constructed with oligonucleotides. After expressed in Escherichia coli, soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%-53% of soluble total proteins) and the purified yield was more than 30 mg/L by one-step purification. Then, the purified AHc was used to vaccinate Balb/c mice, which developed a strong and specific immune response as expected following administration of AHe protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble, stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab, but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.
Assuntos
Animais , Feminino , Camundongos , Anticorpos Antibacterianos , Sangue , Vacinas Bacterianas , Genética , Alergia e Imunologia , Toxinas Botulínicas Tipo A , Genética , Alergia e Imunologia , Botulismo , Alergia e Imunologia , Clostridium botulinum tipo A , Genética , Alergia e Imunologia , Escherichia coli , Genética , Metabolismo , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Genética , Alergia e Imunologia , Linfócitos T , Alergia e Imunologia , Vacinas de DNA , Genética , Alergia e ImunologiaRESUMO
Objective:To determine if suitable electric pulses-mediated DNA and DNA and bupivacaine complexes delivery technologies could enhance effects of botulinum neurotoxin serotype A (BoNT/A) DNA vaccines in mouse model. Methods:Vaccination of mice i.m. with plasmid DNA replicon vaccine pSCARSHc and conventional plasmid DNA vaccine pcDNASHc following electric pulses and with DNA and bupivacaine complexes. AHc-specific group antibody ELISA titers and lymphocyte proliferative responses of mice were detected and IgG1 and IgG2a isotype profiles were assayed. Results:Immune effects of DNA vaccines were enhanced following electric pulses and bupivacaine delivery. Effects of DNA vaccines following electric pulses were better than that of DNA vaccines formulated with bupivacaine,and the combined delivery technology of electric pulses and bupivacaine induced the highest level of specific antibodies and lymphocyte proliferative responses. Plasmid DNA replicon vaccine pSCARSHc induced relatively higher AHc-specific antibodies and lymphocyte proliferative responses in immunized Balb/c mice than conventional plasmid DNA vaccine pcDNASHc in these DNA delivery technologies. And vaccine pSCARSHc induced Th2/Th1-type immune responses with a general bias to Th2-type,and vaccine pcDNASHc induced Th2-type immune responses. Conclusion:Suitable electric pulses-mediated DNA and DNA and bupivacaine complexes delivery technologies could enhance effects of BoNT/A DNA vaccines in mouse model. Therefore,the methods described here potentially provide suitable strategies in developing an efficacious vaccine against botulinum neurotoxin serotype A.
RESUMO
DNA-based replicon expression vector pSMCTA and helper vector pSHCTA were constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with the RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3'-untranslated region (UTR). The RNA polymerase II -dependent cytomegalovirus immediate early (CMV IE) enhancer/promoter and T7 promoter in pSMCTA and pSHCTA could drive transcription to produce replicon RNA in vivo and ex vivo. High level expression of foreign genes (GFP and LacZ) could be demonstrated by transfecting BHK21 cells with the new replicon expression vectors based on both DNA and RNA, and recombinant virus particles (RVP) be prepared by cotranfecting the expression vectors with the helper vectors. Foreign genes were also highly expressed in cells (BHK21) which were infected with RVP activated by alpha-chymotrypsin. The bifunctional replicon vectors can be used in highly efficient expression of foreign genes and preparation of RVP ex vivo, also in development of replicon vaccines and gene therapy vectors in vivo.
Assuntos
Regiões 3' não Traduzidas , Genética , Clonagem Molecular , DNA Viral , Genética , Vetores Genéticos , Genética , RNA Viral , Genética , Proteínas Recombinantes , Genética , Replicon , Genética , Vírus da Floresta de Semliki , Genética , Vírion , Genética , Montagem de Vírus , GenéticaRESUMO
The fusion protein of Humanized mouse anti-human fibrin ScFv and the low molecular weight urokinase (IIn-UK) contained seven disulfide bonds and formed inclusion body while expressing in normal E. coli strain. By coexpressing DsbC and using the special E. coli strain Origami(DE3) which was trxB/gor double mutant, the fusion protein IIn-UK was functionally expressed in the cytoplasm of E. coli. The expressed fusion protein in the soluble fraction was purified by using affinity chromatography specific against urokinase. The purified fusion protein could combine the thrombus in vitro, and the specific activity of urokinase reached 80,000 IU/mg fusion protein. The result showed that the fusion protein retained the activity of two moieties, and this study laid a foundation for further research of targeting thrombolytic agent.