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Objective@#To sequence the 3′UTR of enterovirus 71 strains, investigate its foundation and impact in virulence by constructing a 3′UTR-replaced recombinant cDNA infectious clone.@*Methods@#Viral RNA of EV-A71 isolated viruses were extracted, and the nucleotide analysis was performed after sequencing. The 3′UTR of a full-length infectious clone of SDLY107 strain was replaced by its corresponding part of SDLY1 strain, and then the recombinant virus was constructed and identified.@*Results@#The nine isolated strains were classified into sub-genotype C4a of enterovirus (EV)-A71 by analysis, and nucleotide sequence homology for 3′UTR were 94%-100%. 3′UTR of EV-A71 strains may be associated with its pathogenicity. Identification of the rescued virus by sequencing and indirect immunofluorescence confirmed the successful construction of infectious recombinant virus.@*Conclusions@#Sequence analysis indicated that the 3′UTR may be involved in the pathogenicity of EV-A71. The recombinant virus SDLY107(1-3′UTR) was rescued successfully. Our study may provide evidence for further research on the influence of 3′UTR on the virulence of enterovirus 71.
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Objective@#To explore the influence of pVR-IL15 gene adjuvant on the immune responses of different immunization strategies.@*Methods@#The sequential immunization strategies were used in BALB/c mice with a DNA vaccine, recombinant modified vaccinia virus Ankara and recombinant adenovirus expressing HBsAg respectively and combined with a gene adjuvant pVR-IL15. Cellular and humoral immune responses were evaluated by IFN-γ enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA), respectively.@*Results@#The levels of humoral and cellular immune responses in the immune groups combined with pVR-IL15 were significantly higher than those of the non-combined with pVR-IL15.@*Conclusions@#The pVR-IL15 gene adjuvant can enhance the immune responses induced by the recombinant viruses expressing HBsAg.
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Objective To elucidate the molecular basis on the differences of infectivity in infected cells between Sindbis virus(SINV:YN87448 virus)and Sindbis like virus(SINLV:XJ-160 virus).Methods Compare the E(glycoprotein)gene sequence and secondary structure of YN87448 virus and XJ-160 virus by bioinformatics analysis.Analyze the contribution of E gene to the biological differences between SINV and SINLV by constructing recombinant virus.Results By bioinformatics analysis,YN87448 virus and XJ-160 virus have the same genomic structure,which has 11 717 nt and 11 626 nt respectively.There are 82 amino acid differences between E gene of these two viruses,and showed scattered distribution.The main peak is basically the same for the hydrophobic of the E gene protein,but in some region existing small differences.The recombinant virus which exchanged the E gene of XJ-160 virus with YN87448 virus totally showed the biological character of YN87448 virus,either in the showing time of CPE,plaque forming time and plaque diameter,or in expression of functional proteins.Conclusion E gene plays a major role in the differences of infectivity in infected cells between SINV and SINLV,this result provide the molecular biological evidences for elucidating the biological differences between SINV and SINLV.
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Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.
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The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.
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The design of DNA-based alphavirus vectors significantly improves the utility of these replicon vectors. The DNA-based replicon vectors can be used in expressing foreign genes and preparing RVP in virto efficiently, also in developing replicon vaccines and gene therapy vectors in vivo. The approach involved the conversion a RNA-based replicon vector into a layered DNA-based replicon vector by the RNA polymerase Ⅱ promoter and transcription termination/polyadenylation signal transcribed replicon RNA from DNA. When DNA-based alphavirus vector tranfected into cells, the first layer includes a eukaryotic RNA polymerase Ⅱ expression cassette that initiates transcription of RNA in nucleus. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the RNA vector corresponds to virus RNA replication cycle and results in high level expression of foreign gene. DNA and RNA-based bifunctional replicon expression vector pSCTA and helper vector pSHCTA were successfully constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with CMV promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3′-untranslated region (UTR). In order to obtain DNA-based highly efficient replicon vectors, they were further modified to construct additional three DNA-based SFV replicon expression vectors and corresponding helper vectors. To investigate the efficiency of foreign gene expression level by the four different DNA-based SFV expression vectors and recombinant virus particle (RVP) prepared by cotranfecting with corresponding helper vectors, improved DNA-based replicon vectors pSCAR and pSHCAR derived from SFV were developed. high level protein could be generated using the new vector system by transfecting DNA into BHK21 cells and High titer of RVP produced by cotranfecting with helper vector. Antigen genes were also expressed in cells by the replicon expression vector. Additionally, reporter gene expression was observed in mice muscle following injection with SFV DNA vector. Anti-?-Gal antibody response and cell-mediated immune response were induced after intramuscular inoculation of the ?-Gal-encoding SFV replicon DNA. The results suggested that highly efficient DNA-based replicon vectors pSCAR and pSHCAR were constructed by modifying the SFV vectors. The improved DNA-based replicon vectors enhance the utility of them, and can be developed as potentially replicon vaccines and gene therapy vectors.
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Objective To observe the morphologic features of neurons labeled with green fluorescent protein (GFP) gene recombinant Sindibis virus in the deep part of lamina Ⅲ in the medullary dorsal horn (MDH) of the rat. Methods Neurons were infected and labeled with GFP gene recombinant Sindibis virus injected into lamina Ⅲ.Immunohistchemical staining showed the labeling results.The morphologic features of the GFP labeled neurons were revealed after reconstruction. Results A few GFP-labeled single neurons were found in the distal portions away from the virus injection site within the deep part of lamina Ⅲ of the MDH.According to the morphological features,especially their axons and axonal collaterals,GFP labeled neurons were classified into projection neurons and intrinsic neurons.The axons and their collaterals of the projection neurons entered to the superficial part of lamina Ⅲ,lamina Ⅱ and/or the medullary reticular formation.Most of the axons and their collaterals of the intrinsic neurons extended mainly within lamina Ⅲ.Conclusions According to the morphological features of axons and their axonal collaterals,GFP labeled neurons were classified into projection neurons and intrinsic neurons in the deep part of lamina Ⅲ of the MDH.GFP gene recombinant virus labeling technique is an effective method to investigate the morphological features of neurons.
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The green fluorescence protein (GFP) gene recombinant virus labeling is a new method for the neuroanatomical studies. It is effective to investigate the morphological features of neurons, the chemical architectures (neural active substances and their receptors) of the neurons, the relationships between the neuronal morphological features and their termination areas or their functions, and local circuits related to specific functions. This method remedies the defects of previous morphological methods and could provide direct morphological evidence for the functional investigations.