Construction of vectors expressing bioactive heterodimeric and single-chain murine interleukin-12 for gene therapy.
It has been well demonstrated that interleukin-12 (IL-12) could be useful to defend against a variety of pathogens, to suppress tumor growth and metastasis, and even to be employed as an adjuvant of vaccines to enhance beneficial type 1 T helper (Th1) cell response over detrimental type 2 T helper (Th2) cell responses. To apply IL-12 genes in gene therapy such as a DNA vaccine, a pIL-12 vector was constructed that contained two cytomegalovirus (CMV) promoters to drive the expression of p35 and p40 subunits, respectively. In addition, a pscIL-12 vector was designed with a linker to fuse p35 cDNA with p40 cDNA to produce a single-chain IL-12 protein, ensuring not only that the expression of p35 and p40 subunits was equally expressed, but also that no free p40 subunits interfered with IL-12 activity. The data suggested pIL-12 could produce a rather high level of biologically active IL-12 after transfection of COS cell lines as well as C2C12 muscle cell lines, as measured by both concanavalin A blast proliferation assay and enzyme-linked immunosorbent assay. Interestingly, the pscIL-12 vector could also express a bioactive murine IL-12 fusion protein in vitro. Furthermore, in vivo functional studies also demonstrated that mice co-immunized with a pS vector expressing the major envelope protein of hepatitis B virus (HBV) and IL-12 vectors encoding native IL-12 or single-chain IL-12 fusion protein elicited higher levels of IgG2a anti-HBs antibody and of Th1-related cytokine. Because p35 and p40 genes can be expressed in a vector by using a single promoter, pscIL-12 should be useful in future applications for nucleic acid vaccination or for gene therapy against diseases.
Lee
Y L
YL
Graduate Institute of Microbiology, National Taiwan University, Taipei, Republic of China.
Tao
M H
MH
Chow
Y H
YH
Chiang
B L
BL
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D019281:Dimerization; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006510:Hepatitis B Antibodies / Q000097:blood; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood; D018664:Interleukin-12 / Q000737:chemistry / Q000235:genetics*; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D009132:Muscles / Q000166:cytology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D018417:Th1 Cells / Q000276:immunology; D019444:Vaccines, DNA / Q000235:genetics*
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D018664:Interleukin-12