[Construction, expression and immunogenicity study of a chimeric MS/hIL-12 eukaryotic expression plasmid].
OBJECTIVE
To construct and express a chimeric Mtb8.4 with signal peptide (MS)/hIL12 eukaryotic expression plasmid, and to study the immunogenicity of the MS/hIL-12 chimeric genetic vaccines.


METHODS
The MS/hIL-12 chimeric gene was amplified by polymerase chain reaction (PCR) and cloned into the eukaryotic expression vector pCI-neo. The correct pCI-neo-MS/hIL12 (pMSI) recombinant plasmid was identified by PCR, restricted enzyme digestion and DNA sequencing. COS-7 cells were transfected with pMSI constructs by cationic liposome. After 48 hours, mRNA of the target gene was detected by RT-PCR, and hIL-12 protein in culture supernatant and cell lysates was detected by Western blot. C57BL/6N mice were vaccinated with MS/hIL-12 chimeric gene vaccine for three times at 3 week intervals. Four weeks after the final inoculation, three mice were sacrificed for measurement of the cytokine response and cytotoxic T lymphocyte (CTL) induction.


RESULTS
The accuracy of plasmid construction was confirmed by a number of molecular biological techniques. Transfection of COS-7 cells with plasmids pMSI lead to transient expression of fusion proteins. The IFN-gamma and IL-2 titers were (1,521 +/- 48) ng/L and (755 +/- 41) ng/L in MS/hIL-12 chimeric gene vaccine group, (820 +/- 50) ng/L and (297 +/- 31) ng/L in MS gene vaccine group, (1,487 +/- 40) ng/L and (767 +/- 50) ng/L in BCG group, (121 +/- 16) ng/L and (62 +/- 10) ng/L in vacant vector group, and (48 +/- 16) ng/L and (32 +/- 17) ng/L in PBS group respectively. The levels of IFN-gamma and IL-2 in MS/hIL-12 chimeric gene vaccine group were higher than those of MS gene vaccine group, vacant vector group and PBS group (P < 0.01) and was similar to the BCG group (P > 0.05). The level of IL-4 in BCG group [(91 +/- 11) ng/L] increased significantly as compared to other groups (P < 0.01). When effector-cell-to-target-cell ratio (E:T ratio) were 100:1, 50:1, and 10:1 respectively, the CTL activity was 77.5%, 51.2%, 30.3% in MS/hIL-12 chimeric gene vaccine group, 56.2%, 37.8%, 11.5% in MS gene vaccine group, 28.9%, 21.4%, 9.8% in BCG group. The cytotoxicity in MS/hIL-12 chimeric gene vaccine group was higher than that of other groups (P < 0.01).


CONCLUSIONS
When used to construct the chimeric gene vaccine, hIL-12 could improve the immunogenicity of MS gene vaccine.
Li
Hui
H
Department of Infectious Disease, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, China.
Li
Rong
R
Zhong
Sen
S
Ren
Hong
H
Deng
Cun-liang
CL
Shi
Xiao-ling
XL
Wang
Ming-yong
MY
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D005822:Genetic Vectors; D018664:Interleukin-12 / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000276:immunology; D010957:Plasmids; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D032581:Tuberculosis Vaccines / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D032581:Tuberculosis Vaccines; D014614:Vaccines, Synthetic; D018664:Interleukin-12