Cloning and characterization of the heavy chain variable region gene of monoclonal antibody 2F2 against Japanese encephalitis virus / 中国医师杂志

Objective To obtain the heavy chain variable region (VH) gene of monoclonal antibody 2F2 against Japanese encephalitis virus (JEV).Methods Total RNA was isolated with Trizol from hybridoma 2F2 cells,and cDNA of VH was amplified with reverse transcription polymerase chain reaction (RT-PCR) and sequenced.The putative VH gene was expressed in E.coli,and the expressed products was detected with enzyme linked immunosorbent assay (ELISA) to determine the activity to bind JEV.Results The VH gene was 354 bp in length which encodes 118 amino acids.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the VH gene was successfully expressed and purified from inclusion bodies.ELISA result also demonstrated that VH gene expressed products bind purified JEV.Conclusions The VH gene of monoclonal antibody 2F2 against JEV had been cloned.

Construction and Expression of Recombinant Plasmid Encoding prME Protein Derived from Japanese Encephalitis Virus and Granulocyte-macrophage Colony-stimulating Factor of BALB/c Murine / 中国医科大学学报

Objective Construction and expression of recombinant plasmid fusing encoding prME protein derived from Japanese encephalitis virus （JEV） and GM-CSF of BALB/c mouse. Methods GM-CSF gene was ampli/ied by nested-RT-PCR from BALB/c spleen cells. JEV prME protein gene was eluted by the digestion with restrict/on endonucleases BamH I/EcoR I from pJME piasmid. Genetic fusion of prME protein and GM-CSF are subcloned info pcDNA3.1 （＋） eukaryotic vector, and named as pJME/GM-CSF.The recombinant was confirmed by restriction enzymes digestion and DNA sequencing, and then transfected into China hamster ovary （CHO） cells by Lipofectamine2000. pJME/ GM-CSF expression in CHO cells was examined by Western blot. Results We observed 2001 bp and 2472 bp DNA fragments when pJME/GM-CSF was digested with BamHI/EcoRI and BamHI/NotI respectively as expected. The estimated molecular weight of the fusion protein was 85kD. Conclusion The recombinant pJME/GM-CSF was constructed and transfected into CHO cells successfully with pJME/GMCSF stably expressed.

Preliminary application of antibody-capture ELISA detection the antibody of Japanese encephalitis virus / 中国地方病学杂志

Objective To set up an antibody-capture ELISA method to detect the Japanese encephalitis virus(JEV)antibody.Methods ELISA plate was coated with the monoclonal antibody which was specific to the envelope protein epitope E39 of JEV,JEV SA14-14-2 strain as the source of antigen was used to absorb the monoclonal antibody,the absorbed virus used to capture the JEV'S antibody.The antibody that captured ELISA was established.The indirect ELISA method using the virus particles from cell culture was compared with coating ELISA plate,105 clinical serum were checked.Results The background in indirect ELISA assay could not be abscised,positive and negative serum diluted in a ratio of 1:10,1:100,1:1000,the relative value of A posative/A negative were 1.02,0.99,1.13,all<2.1.But the antibody-captured ELISA method when the serum dilution was 1:10,1:100,the A posative/A negative were 3.57,2.94,all>2.1;when the dilution was 1:1000,the A posative/A negative was 1.42,<2.1,it meant the method could distinguish the positive and negative serum efficiently when the dilution Was 1:100,the background problem in indirect ELISA assay could be solved.Antibody-capture method was used to check 105 serum samples,the A posative/A negative over a range of 0.257～0.321(0.262±0.050),all<2.1,no positive sample found.Conclusion The antibody-capture ELISA method has been preliminary set up with a high specificity,capable of quickly identifying JEV from other virus.

Adjuvant effect of granulocyte-macrophage colony-stimulating factor coding gene on cellular immunity of Japanese encephalitis virus DNA vaccine / 中华传染病杂志

Objective To study the adjuvant effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) coding gene on cellular immunity induced by Japanese encephalitis (JE) virus DNA vaccine. Methods GM-CSF coding gene was amplified by nested-reverse transcriptase-polymerase chain reaction (RT-PCR) technique from BALB/c murine spleen cells. Recombinant plasmids pJME/GM-CSF and pGM-CSF were constructed by JE virus (JEV) prM-E protein with GM-CSF coding gene or GM-CSF coding gene only, respectively. The plasmids were transfected into China hamster ovary (CHO) cells by Lipofectamine 2000. The coding protein expressions and distributions were detected by immunofluorescence. The BALB/c mice were vaccinated with indicated immunogens with or without GM-CSF gene. The changes of T lymphocyte subsets in the spleen and levels of intracellular cytokines, such as interferon (IFN)-γ and interleukin (IL)-4 of splenic cells from mice immunized with different immunogens were evaluated by flow cytometry. The cytotoxicity T lymphocyte (CTL) activity was assessed by lactate dehydrogenase (LDH). The data were compared by one-factor analysis of variance and least significant difference. Results The constructed recombinant pGM-CSF and pJME/GM-CSF were confirmed by restrict enzyme digestion and DNA sequencing. The expressions of the above proteins were mainly in the cytoplasm and minor on cell membrane. The percentage of CD4+ T lymphocytes in pJME/GM-CSF vaccinated group was (33.90±0.79)%, which was significantly higher than that of in other groups (t values were 9. 818, 6. 804, 6.594, 10.061, 9.380, and 17.675, all P<0.05). The percentages of CD4+T lymphocytes in pJME +pGM-CSF (0) and pJME+pGM-CSF (-3) vaccinated groups were (29.83±0.61)% and (29.70±0.51)%, respectively, which were both higher than that in pJME+pGM-CSF (+3) vaccinated group of (27.69+0.50)% (t=3.466, t=3.255, both P<0.05). The percentages of CD8+ T cells in pJME/GM-CSF and pJME+pGM-CSF vaccinated groups were both higher than that in empty vector (pcDNA 3.1+) group and JE inactivated vaccine vaccinated group (t values were 3.811, 2.627, 10.537, and 3.811, all P<0.05). The CTL activity in pJME/GM-CSF vaccinated group was (51.48±0.10)%, which was higher than those in other groups (t values were 22.868, 13.823, 5.377, 32.287, 34.632, and 53.795, all P<0.05). The IFN-γ/IL-4 ratios in pJME/GM-CSF, pJME+pGM-CSF (0) and pJME + pGM-CSF (-3) vaccinated groups were (19.13±1.36), (12.32±0.82) and (7.05±0.43), respectively, which were higher than those in other groups (P<0.05). Conclusion GM-CSF coding gene could enhance the cellular immune response induced by Japanese encephalitis DNA vaccine.

Comparison of gene expression and DNA immunization efficacy encoding prME and E proteins derived from Japanese encephalitis virus / 中华传染病杂志

3 ?g/well.All BALB/c mice immunized with pJME by im.and gene gun injection,and JE inactivated vaccines by ip.injection survived for 21 days.BALB/c mice immunized with pJE by im.injection and gene gun injection partially survived for 21 days.Titres of neutralization antibody produced with pJME were higher than pJE.Protective immunity and titre of neutralization antibody produced by im injection was the same as gene gun injection(im/gene gun injection: 1∶320/1∶320) at day 21.The antibody from BALB/c mice sera after twice pJME immunization only reacts with JEV-E protein. Conclusions Expression efficacy of proteins encoded by pJME and pJE in transfected cells is different.Expression level of related proteins was dependant on recombinant amount for transfection in a certain degree.Immunity effect induced with pJME was higher than pJE.The efficacy of DNA immunization produced by gene gun injection was higher than im.injection.Titres of neutralization antibody induced by DNA immunization were correlated to efficacy of protective immunity.Neutralization antibody from BALB/c mice sera produced by pJME immunization contained anti-E antibody against JEV-E protein.

Gene cloning and expression of Japanese encephalitis virus E protein in Escherichia coli / 中华传染病杂志

Objective To express Japanese encephalitis virus (JEV) E protein in Escherichia coli. Methods The gene coding for JEV E protein was amplified by using RT-PCR, and cloned into plasmid pET-28a. The constructed plasmid was transformed into E.coli BL21 (DE3). Expression of E protein by the transformants was induced by IPTG and analyzed by SDS-PAGE and Western blot. Results Five nucleotide changes leading to amino substitutions were identify in the E protein gene of our laboratory strain of JEV when compared to a published gene sequence of JEV E protein. The yield of expressed JEV E protein with relative molecular mass approximately 52?10 3 was 35% of total bacterial proteins. Conclusions JEV E protein was expressed successfully in E.coli, which should be useful for the production of diagnostic reagents and the analysis of gene structure/function of the E protein.

Detection of main flaviviruses using polymerase chain reaction and microwell plate hybridization / 解放军医学杂志

Objective To establish a specific,sensitive and applied method for the detection and differentiation of dengue virus types Ⅰ-Ⅳ,Japanese encephalitis virus and yellow fever virus.Methods Based on the genomes sequence analysis,6 pairs of primers were designed.The special capture probes of dengue virus types Ⅰ-Ⅳ,Japanese encephalitis virus and yellow fever virus were amplified,cloned and sequenced.Then the microwell plates were precoated using these capture probes,and the forward primers were labeled using biotin.The samples were then amplified using the biotin labeled forward primers and reward primers.The microwell plate hybridization was processed for detecting and differentiating the virus.The precoated DNA concentration,precoated time,hybridization temperature and hybridization time were optimized carefully.Results The A value of positive samples were over 0.5,while the average A value of the negative samples was less than 0.1.The S/N value exceeded 10.0.Sensitivity experiment suggested the method of PCR-ELISA could detect the virus RNA in 107 times dilution,while RT-PCR could detect the virus RNA in only 106 times dilution.The stability experiment of PCR-ELISA using DVⅠ suggested that the within-batch coefficient of variation was 6.21%,the between-batch coefficient of variation was 9.92%;the within-batch coefficient of variation in negative control was 1.92%,and the between-batch coefficient of variation in negative control was 3.68%.No visible changes were found on the performance of the coated microwell plates when stored in 4℃for 6 monthes.Conclusion PCR-ELISA is a more sensitive and specific method than RT-PCR is in the early detection and type identification of dengue Ⅰ-Ⅳ types virus,Japanese encephalitis virus and yellow fever virus.