CFP10, ESAT6, Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the key immunodominant antigens of Mycobacterium tuberculosis. In order to construct a eukaryotic vector able to co-express the four genes in one vector, we amplified the target gene fragments encoding the CFP10, ESAT6, Ag85A and Ag85B antigens and inserted them into the multicloning site of the shuttle plasmid vector pcDNA3.1 (+), of which the CFP10 and ESAT6 encoding genes were in frame fused with a linker encoding (Gly4Ser)3 residue, before the fused gene was inserted downstream of CMV promoter with a bovine growth hormone poly A(BGH pA) sequence at the 3'-end; Ag85A and Ag85B encoding genes were fused with a separation of internal ribosome entry site (IRES) sequence before the fused gene cassette was inserted downstream of RSV promoter with a BGH pA sequence at the 3'-end. The final plasmid containing all four genes was confirmed by sequence analysis and designated as pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB). In order to verify the ability of this construct to express target proteins, we then transfected the recombinant plasmid into Human embryonic kidney (HEK) 293T cells and harvested the cell lysates, and the cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis 48 h after transfection. All four of the target proteins were detected in the cell lysates against the respective specific antibodies, suggesting that we have successfully constructed a eukaryotic vector co-expressing the four immunodominant antigens of Mycobacterium tuberculosis, which lay a foundation for the further study of the immunogenicity and protective activity of the four antigens.
Acyltransferases ; biosynthesis ; Antigens, Bacterial ; biosynthesis ; Bacterial Proteins ; biosynthesis ; Genetic Vectors ; HEK293 Cells ; Humans ; Immunodominant Epitopes ; Mycobacterium tuberculosis ; Plasmids

Objective To gain more pathophysiolgic knowledge about acute obstructive hydrocephalus and to explore its rapid and effective treatment by establishing canine acute obstructive hydrocephalus model.Methods Acute obstructive hydrocephalus model was established by injecting cyan-acrylic gel glue into the fourth ventricle via posterior fosse craniotomy in 9 male adult mongrel dogs.At the same time,lateral ventricle catheterization were performed and were fixed on the scalp to connect reservoir bag so that the changes of intracranial pressure (ICP) could be measured dynamically,and the changes of neurological function were observed.Results Acute obstructive hydrocephalus model was successfully established in 6 of the total 9dogs.ICP was (48.2 ± 6.1 ) cm H2 O at 48 hours after the injection and was (56.4 ± 5.7 ) cm H2 O at 72 hours after the injection,it increased 392％ and 459 ％ respectively.And the ICP after injection was significantly different(P ＜ 0.01 )compared with that before injection (12.3 ± 3.1 )cm H2O.Conclusion The establishment of acute obstructive hydrocephalus model has high success rate,and is easy to reduplicate; ICP could be measured dynamically and also could be reduced by releasing CSF;Thus,ventricular drainage is the most rapid and effective treatment for acute obstructive hydrocephalus.

Tuberculosis (TB) remains an enormous health burden worldwide.To date,Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the unique anti-TB vaccine available for humans,which provides an important but limited protection from the Mycobacterium tuberculosis (Mtb) infection.It is therefore an urgent need to develop better vaccines and vaccination strategies to prevent the spread of Mtb infection.Heterologous prime-boost vaccination strategies using both BCG and novel anti-TB vaccines have been demonstrated to induce robust immune responses than BCG alone.We have previously demonstrated that a recombinant adenoviral vector Ad5-CEAB co-expressing CFP10,ESAT6,Ag85A and Ag85B of Mtb was able to induce robust antigen-specific immune responses in mice.In the present study,we examined immunological effects of Ad5-CEAB in the mice primed with BCG and boosted with a single dose of the virus via an intranasal route.Results demonstrated that this vaccination strategy could effectively induce strong antigen-specific mucosal and humoral immune responses.These immune responses were characterized with an increased productions of cytokines (IL-12 and IFN-γ),increased concentration of secretary IgA (sIgA) in bronchoalveolar lavage fluid (BALF) and serum IgG in mice in comparison with mice in BCG group.These data suggested that the regimen of BCG prime-single dose of Ad5-CEAB boosted strategy was novel for inducing antigen-specific immune responses in response to Mtb antigens in vivo,which warrants for further development of adenoviral-based vaccine against Mtb infection.