ABSTRACT
<p><b>OBJECTIVE</b>To establish a loop-mediated isothermal amplification (LAMP) method for rapid diagnosis of Vibrio cholerae.</p><p><b>METHODS</b>Based on the ompW nucleic sequence of Vibrio cholerae, a pair of primers was designed for LAMP. The reaction conditions were optimized, and the specificity, sensitivity, and practicability of LAMP were tested using 47 bacterial strains and simulated contaminated sites.</p><p><b>RESULTS</b>The results of viable bacterium count showed that LAMP was capable of detecting Vibrio cholerae at a level as low as 1.6x10(2) cfu/ml. The minimal detectable concentration was 1.6+10(3) cfu/ml for simulated contaminated samples such as feces and seawater, and 1.6+10(4) cfu/ml for contaminated milk. All the 21 strains of Vibrio cholerae yielded positive results in LAMP, and the 26 strains of other bacteria all showed negative results, with a detection specificity of 100%.</p><p><b>CONCLUSION</b>The established LAMP method has high specificity and sensitivity for detecting Vibrio cholerae and is applicable in field monitoring and epidemiological study of Vibrio cholerae.</p>
Subject(s)
Humans , Bacterial Proteins , Genetics , Cholera , Diagnosis , Microbiology , Clinical Laboratory Techniques , Methods , Nucleic Acid Amplification Techniques , Methods , Sensitivity and Specificity , Vibrio cholerae , GeneticsABSTRACT
Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.
ABSTRACT
Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.
ABSTRACT
Objective:To construct a DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the anti-HBV siRNA genes, and to express this DNA vaccine in HepG2 cells. Methods:The HBV multi-epitope antigen gene was designed and synthesized before it was fused with enhanced green fluorescent protein(EGFP) gene, and cloned into the multi-clone site(MCS) of the eukaryotic expression vector pVAX1. The expressinig units of hIL-12 and siRNA were cloned into the BspH I and Mlu I site of pVAX1 respectively. Then the recombinant plasmid pVAX1-siHBV-HB-EGFP-hIL12 was transiently transfected HepG2 cells. The expression of HBV compound multi-epitope gene was observed through EGFP report gene. The expression of hIL-12 was analyzed by ELISA and the effects of anti-HBV siRNA was confirmed with rtPCR . Results: The analysis of enzyme digestion and sequencing both demonstrated that the trible-expressing HBV DNA vaccine has been constructed successfully. The green fluorescent image was detected in the transfected cells which could confirm the expression of the multi-epitope antigen gene. The amount of hIL-12 secretion was 1289pg/ml in supernatant at 48h after transfection and 1712pg/ml at 72h after transfection. The mRNA amount of HBV S gene, which was the siRNA target, had been obviously knockdown. Conclusion: The DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the siRNA genes was constructed and transiently expressed in HepG2 cells, and siRNA had shown us a good anti-HBV effect. It laid a foundation of further study on anti-HBV effect of the new DNA vaccine.
ABSTRACT
Objective:A novel tuberculosis DNA vaccine integrating siRNA,fusional antigen and hIL-12 triple expression units was constructed in our laboratory.Methods:To evaluate the independent expression of the three expression units,two eukaryotic expression plasmid pVAX1-siRNA-PVAE[EGFP]-hIL12 with TB fusional gene Ag85B-ESAT6(PVAE) and enhanced green fluorescent protein(EGFP),and pVAX1-siEGFP-PVAE[EGFP]-hIL12 with siRNA coding sequence targeting EGFP instead of Mcl-1L were constructed.Then two plasmids were used to transfer human embryonic kidney 293 cells.Based on EGFP report gene,it was demonstrated that siRNA expression unit and fusional antigen gene were independently expressed.Results:The hIL12 expression at 48h and 72h post transfection were also detected by ELISA analysis up to 1571.63pg/ml and 2392.25pg/ml respectively in the cell culture fluid.Conclusion:The results demonstrated that the novel DNA vaccine with siRNA,TB fusional antigen and hIL12 three expression units in the same plasmid frame is successfully constructed and independently expressed in eukaryotic cells.It laid a foundation for further animal model study on anti-tuberculosis effects of this novel DNA vaccine.
ABSTRACT
Hepatitis C virus(HCV)is the major etiology of non-A,non-B hepatitis.At present,neither a vaccine nor any effective therapy is available.Multi-epitope DNA vaccine(minigenes/epigenes)is a novel nucleic acid vaccine which can induces high effective cellular and humoral immune responses and has good perspective in clearing Hepatitis C virus through screening and assembling optimal antigen epitope genes(T cell、B cell epitope included).It is advanced in covering more HCV subtypes,inducing comprehensive anti-HCV immune responses and reducing the negative influence caused by irrelevant,disturbing and suppressive sequence as far as possible through selecting the most potential protective epitopes in DNA vaccine design.The recent research progress on HCV compound multi-epitope DNA vaccine and future prospects were reviewed.