Abstract:

Objective To preliminarily investigate the expression of long-chain non-coding RNA 017418 (lncRNA 017418) and cytokines during immune response of mice induced by recombinant protein P29 (rP29).Methods The recombinant fusion expression strain of Escherichia coli pET28a-P29/BL21 was incubated with LB liquid medium, to which isopropyl-β-D-thioglycoside (IPTG) was added to induce protein expression. The purified recombinant protein rP29 was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thirty-six female BALB/c mice were randomly divided into the control group, adjuvant group and immunization group (12 mice each). To immunization group mice, 10 μg purified rP29 was mixed with an equal volume of Freund’s complete adjuvant forming emulsion, and injected subcutaneously at three points at abdomen at a total volume of 100 μl/mouse. The adjuvant group was injected with an equal volume of Freund’s complete adjuvant alone, while the control group received no treatment. Two weeks after the first immunization, the same volume of booster with incomplete Freund’s adjuvant was given once. Two weeks after boosting, spleen cell suspensions were prepared in sterile condition and lymphocytes were separated. Flow cytometry was performed to assay the lymphocyte subsets CD4 ⁺ T, CD8 ⁺ T and B lymphocytes. The expression of lncRNA 017418, Notch1, interleukin-2 (IL-2), interferon gamma (IFN-γ) and IL-10 in lymphocytes was detected by real-time fluorescence quantitative PCR (qRT-PCR). Data were analyzed with SPSS 17.0 and GraphPad Prism 6.0 softwares. Results Produced by IPTG induction and affinity chromatography purification, the purified rP29 showed a relative molecular weight of ~31 000. qRT-PCR revealed that the relative expression of lncRNA 017418 in spleen lymphocytes in the immunization group was 2.25 ± 0.10, which was significantly higher than those in the control (0.81 ± 0.05) and adjuvant groups (0.99 ± 0.13) (P < 0.01). The proportions of CD4 ⁺ T, CD8 ⁺ T and B lymphocytes found by flow cytometry were 22.0%, 9.2% and 59.8% in the control group; 22.8%, 7.6% and 60.7%in the adjuvant group; and 22.1%, 9.7% and 60.4% in the immunization group, respectively. The relative expression of lncRNA 017418 in CD4 ⁺ T lymphocyte in the immunization group was 1.49 ± 0.03, significantly higher than those in the control group (0.97 ± 0.02) and the adjuvant group (1.06 ± 0.10) (P < 0.01); those in the CD8 ⁺ T cells in the three groups were 1.87 ± 0.12, 2.06 ± 0.14, and 2.00 ± 0.09, respectively (P > 0.05); and those in the B cells were 1.03 ± 0.03, 0.97 ± 0.07, and 1.08 ± 0.12, respectively (P > 0.05). The relative expression of Notch1 in the CD4 ⁺ T lymphocytes was 1.92 ± 0.04 in the immunization group, higher than those in the control group (1.10 ± 0.10) and adjuvant group (1.20 ± 0.08) (P < 0.01). The relative expression of IL-2 was 1.45 ± 0.07 in the immunization group, higher than those in the control group (1.07 ± 0.08) and adjuvant group (1.09 ± 0.118) (P < 0.01). The relative expression of IFN-γ was 1.51 ± 0.11 in the immunization group, higher than those in the control group (0.92 ± 0.05) and adjuvant group (1.03 ± 0.14) (P < 0.01, P < 0.05). The relative expression of IL-10 was 0.52 ± 0.01 in the immunization group, lower than those in the control group (0.95 ± 0.04) and adjuvant group (1.05 ± 0.10) (P < 0.01). There is no statistically significant difference found between the control group and the adjuvant group(P > 0.05). Pearson correlation coefficient analysis showed that lncRNA 017418 was positively correlated with Notch1, IL-2, and IFN-γ (r = 0.824 4, 0.590 2, 0.841 5; P < 0.01), and negatively correlated with IL-10 (r = -0.443 2; P < 0.05). Conclusion LncRNA 017418 expression is up-regulated in rP29-immunized mice; the expression of Notch1, IL-2 and IFN-γ are up-regulated in CD4 ⁺ T lymphocytes; and IL-10 expression is down-regulated in CD4 ⁺ T lymphocyte.

Key words: Recombinant protein P29, CD4 ⁺ T lymphocyte, Long non-coding RNA 017418, Immunoprotection