[Immunogenicity of chimeric gene vaccine Mtb8.4/hIL12].
OBJECTIVE
To construct chimeric gene vaccine Mtb8.4/hIL-12, express it in COS-7 cells and study its immunogenicity.


METHODS
Chimeric gene Mtb8.4/hIL-12 was amplified by PCR and cloned into the eukaryotic vector pCI-neo to construct the recombinant plasmid pCI-neo-Mtb8.4/hIL12. After the recombinant plasmid was identified by restriction enzyme digestion analysis, PCR and DNA sequencing, COS-7 cells were transfected with pCI-neo-Mtb8.4/hIL12 through cationic liposome. 48 hours later, the expression of mRNA was detected by RT-PCR and the level of hIL-12 in culture supernatant and cell lysates were detected by Western blot. C57BL/6N mice were vaccinated with chimeric gene vaccine Mtb8.4/hIL-12 three times at the interval of 3 weeks each time. Four weeks after the final inoculation, three mice were sacrificed to assess the cytotoxicity of CTLs and response to cytokine.


RESULTS
The recombinant plasmid pCI-neo-Mtb8.4/hIL12 was constructed successfully. After COS-7 cells were transfected with pCI-neo-Mtb8.4/hIL12, chimeric gene Mtb8.4/hIL12 was expressed in COS-7 cells. The chimeric gene vaccine could induce strong antigen-specific immune response. With the increase of IFN-gamma and IL-2 secretion and the decrease of IL-4 secretion, the cytotoxicity of specific CTLs was heightened.


CONCLUSIONS
Recombinant plasmid pCI-neo-Mtb8.4/hIL12 has been successfully constructed and expressed in COS-7 cells. The constructed chimeric gene vaccine Mtb8.4/hIL12 is of strong immunogenicity and can obviously induce the cytotoxicity of CTLs.
Li
Hui
H
Department of Infectious Diseases, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, China. lihui_luzhou@163.com
Li
Rong
R
Zhong
Sen
S
Luo
Yue-bei
YB
Ren
Hong
H
Deng
Cun-liang
CL
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics*; D019677:Artificial Gene Fusion / Q000379:methods*; D001426:Bacterial Proteins / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018664:Interleukin-12 / Q000032:analysis / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014162:Transfection; D019444:Vaccines, DNA / Q000235:genetics* / Q000276:immunology*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016207:Cytokines; C114074:Mtb8.4 protein, Mycobacterium tuberculosis; D019444:Vaccines, DNA; D018664:Interleukin-12; D012313:RNA