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.

Objective To study the effect of lgG Fc gene on JEV DNA vaccine immunity. Methods Gene encoding IgG Fc was amplified by nested-RT-PCR technique from BALB/c murine spleen cells. JEV prME protein gene was obtained with restriction endonuclease BamH Ⅰ/EcoR Ⅰ from the eukaryotic recombinant named after pJME, which was constructed by us before. Recombinant, named after pJME/IgG Fc, with above two genes encoding JEV prME protein and BALB/c murine IgG Fc was constructed, and was tested by restriction enzymes analysis and DNA sequencing, then was transfected into China hamster ovary (CHO) cells by Lipo-fectAMINE 2000. Distribution and expression of the fusion proteins encoded by JEV prME protein and BALB/c murine IgG Fc genes in transfected CHO cells were detected by immunofluorescence and Western blot. The BALB/c micc were vaccinated with pJME/IgG Fc via intramuscular injection. Then the cytotoxic T lymphocyt (CTL) activity were assessed by lactic dehydrogenase (LDH) and the neutralizing antibody titer were assessed by 80% plaque reduction neutralization test. Results Molecular weights (2001 bp, 2730 bp) of the two in- serts released from pJME/IgG Fc with two group of restriction analysis associated with BamH 1/EcoR I and BamH Ⅰ/Not Ⅰ were correlated to the expected theoretic results respectively. It was estimated that molecular weight (Mr) of the fusion protein was 101 x 103. The expression of the above fusion protein was mainly distribu- ted in endochylema of transfected CHO cells,and not much in membrane of transfected CHO cells. CHO cells transfected with pJME/IgG Fc could express the fusion protein at the 32th cell passage. After immunization, the CTL activity and the neutralizing antibody titer in the pJMF/IgG Fc vaccinated group increased significantly compared with other vaccinated groups(P <0.05). Conclusion The recombinant pJME/IgG Fc was construc- ted and transfected into CHO cells successfully, and CHO cellular lines expressed fusion protein encoded by JEV prME protein and BALB/c murine lgG Fc genes stably were obtained. IgG Fc gene could reinforce the cellular immunity and humoral immunity of JEV DNA vaccine.

The multifunctional member for improvement of the active tourniquet adopts data transmission module and GPS receiver, which can be a accessory of the tourniquet. The member has such functions as time alarm, identification and locating of the casualty.

AIM: To appropriately modify the defects of active tourniquets in order to act as various tourniquet attachments in Chinese Army.METHODS: This attachment is classified into individual-solider end and searching-rescue end, and data at both ends are collected and transmitted by using wireless data transmission module. Individual-solider end is composed of timing alarm circuit, bit display tube and GPS module, etc. and the data are processed by using single chip of wireless data transmission module. Searching-rescue end adopts the same data transmission module as individual-solider end, and port connects to computer in order to realize two-dimensional data delivery and communication to individual-solider end by using computer-controlled software, i.e. battleground immediate pursuit rescue system.RESULTS: As various tourniquet attachments, individual-solider end is characterized by timing alarm function in the phase of installing tourniquet. Necessarily, we may start searching-rescue function, and the matching with searching-rescue end may realize personal identification and localization of wounded soldiers.CONCLUSION: As a common tourniquet attachment, it is provided with installing alarm, personal identification and localization of wounded soldiers; however, its function is still thin. It brings a lot of evidences for further improvement through installing a physiological probe at individual-solider end to collect physiological data and distantly monitor real-time vital signs of wounded soldiers.

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.