Expression Improvement of Recombinant Plasmids of the Interleukin-7 Gene in Chitosan-Derived Nanoparticles and Their Elevation of Mice Immunity

Abstract

To investigate a safe and effective approach for enhancing the in vivo expression of recombinant genes and improving the systemic immunity of animals against infectious diseases, we employed the interleukin-7 (IL-7) gene from Tibetan pigs to construct a recombinant eukaryotic plasmid (VRTPIL-7). We first examined VRTPIL-7's bioactivity on porcine lymphocytes in vitro and then encapsulated it with polyethylenimine (PEI), chitosan copolymer (CS), PEG-modified galactosylated chitosan (CS-PEG-GAL) and methoxy poly (ethylene glycol) (PEG) and PEI-modified CS (CS-PEG-PEI) nanoparticles using the ionotropic gelation technique. Next, we intramuscularly or intraperitoneally injected mice with various nanoparticles containing VRTPIL-7 to evaluate their immunoregulatory effects in vivo. We observed a significant increase in neutralizing antibodies and specific IgG levels in response to the rabies vaccine in the treated mice compared to the controls. Treated mice also exhibited increased leukocytes, CD8+ and CD4+ T lymphocytes, and elevated mRNA levels of toll-like receptors (TLR1/4/6/9), IL-1, IL-2, IL-4, IL-6, IL-7, IL-23, and transforming growth factor-beta (TGF-β). Notably, the recombinant IL-7 gene encapsulated in CS-PEG-PEI induced the highest levels of immunoglobulins, CD4+ and CD8+ T cells, TLRs, and cytokines in the mice's blood, suggesting that chitosan-PEG-PEI may be a promising carrier for in vivo IL-7 gene expression and enhanced innate and adaptive immunity for the prevention of animal diseases.

Keywords: chitosan derivatives; gene expression; immunity; mice; nanoparticle; pig interleukin-7.

Figure 1

The map of entrapped VTPIL-7 nanoparticles size. (A) VTPIL-7-CS; (B) VTPIL-7-PEI; (C) VTPIL-7-CS-PEG-Lac; (D) VTPIL-7-CS-PEI-PEG.

Figure 2

The map of entrapped VTPIL-7 nanoparticles Zeta Potential (mV). (A) VTPIL-7-CS; (B) VTPIL-7-PEI; (C) VTPIL-7-CS-PEG-Lac; (D) VTPIL-7-CS-PEI-PEG.

Figure 3

Electrophoresis of RT-PCR products of the samples of cells transfected with different nanoparticles (2% agarose gel). Lane1: RT-PCR product of VTPIL-7-CS, Lane2: RT-PCR product of VTPIL-7-PEI, Lane3: RT-PCR product of VTPIL-7-CS-PEG-Lac, Lane 4: RT-PCR product of VTPIL-7-CS-PEI-PEG, Lane 5: RT-PCR product of VR1020-CS, M: DNA Marker.

Figure 4

The lymphoblasts proliferation analyzed using a Cell Counting Kit-8 (CCK8) 12–72 h post-inoculation by the supernatant of HEK-293 cells transfected by chitosan-wrapped VTPIL-7s and chitosan-blank plasmid controls. Statistical significance was shown by letter symbols. Different letters above the bars of the groups indicate significant differences at p < 0.05. The same letters notated above the bars indicate that the differences among the groups were not significant. The followings are all the same as here.

Figure 5

The changes of leukocytes (a), erythrocytes (b), platelets (c), and hemoglobin (d) in the peripheral blood and weight (e) of VTPIL-7-CS, VTPIL-7-CS-PEG-PEI and VTPIL-7-CS-PEG-Lac treated mice and control groups. Statistical significance was shown by letter symbols. Different letters above the bars of the groups indicate significant differences at p < 0.05. The same letters notated above the bars indicate that the differences among the groups were not significant.

Figure 6

The variation of CD4+ and CD8+ T cells and their ratio in peripheral blood of mice treated with VTPIL-7-CS, VTPIL-7-CS-PEG-PEI, VTPIL-7-CS-PEG-Lac, and plasmid controls. (a) CD4+ T cell level (%); (b) CD8+ T cell level (%); (c) The ratio of CD4+/CD8+ T cell. Statistical significance was shown by letter symbols. Different letters above the bars of the groups indicate significant differences at p < 0.05. The same letters notated above the bars indicate that the differences among the groups were not significant.

Figure 7

The IgG1, IgG2a, IgG, and their ratio in the sera of mice inoculated with VTPIL-7-CS, VTPIL-7-CS-PEG-PEI, VTPIL-7-CS-PEG-Lac, and plasmid controls was quantified by ELISA. (a) IgG level; (b) IgG1 level; (c) IgG2a level; (d) The ratio of IgG1/IgG2a. Statistical significance was shown by letter symbols. Different letters above the bars of the groups indicate significant differences at p < 0.05. The same letters notated above the bars indicate that the differences among the groups were not significant.

Figure 8

The expression levels of four toll-like receptors (TLRs) at the gene level in inoculated mice analyzed by RT-qPCR. (a) The expression level of TLR1; (b) The expression level of TLR4; (c) The expression level of TLR6; (d) The expression level of TLR9. Statistical significance was shown by letter symbols. Different letters above the bars of the groups indicate significant differences at p < 0.05. The same letters notated above the bars indicate that the differences among them were not significant.

Figure 9

The expression levels of immunoregulatory cytokines at the gene level in inoculated mice analyzed by RT-qPCR. (a) TGF-β expression level; (b) IL-1 expression levels; (c) IL-2 expression level; (d) IL-4 expression level; (e) IL-6 expression level; (f) IL-7 expression level; (g) IL-23 expression level. Statistical significance was shown by letter displays. Different letters above the bars indicate significant differences at p < 0.05. The same letters notated above the bars indicate that the differences were not significant.

Figure 10

The changes in weights of the experimental mice after Rabie vaccination (n = 10).

Figure 11

The changes of neutralizing antibodies (a) and specific IgG (b) in the sera of the experimental mice after Rabie vaccination (n = 10). ^★: p < 0.05.