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观察真核重组表达质粒pFlt3L及pCCL5对携带HBc抗原的DNA疫苗诱导的抗原特异性免疫应答的促进作用。将pFlt3L及pCCL5分别用脂质体的方法转染Hep G2细胞,然后采用骨髓细胞增殖及趋化小室实验检测细胞上清Flt3L及CCL5两种细胞因子的生物学活性。将pFlt3L和pCCL5两种重组质粒单独或联合使用与携带HBc抗原的DNA疫苗经肌内注射法免疫小鼠,采用MTT法检测脾淋巴细胞增殖、流式细胞仪检测脾CD8+T淋巴细胞中IFN-γ表达、ELISA法检测脾淋巴细胞培养上清IL-4含量及乳酸脱氢酶(LDH)释放法检测特异性CTL杀伤活性。结果:骨髓细胞增殖及趋化实验证实了Flt3L及CCL5均有生物学活性。pFlt3L和pCCL5单独或联合使用均可促进特异性淋巴细胞增殖反应(P<0.05),提高小鼠脾脏CD8+T淋巴细胞中IFN-γ表达量(P<0.05或P<0.01),IL-4表达水平在各组无显著区别(P>0.05),Flt3L+CCL5组小鼠脾细胞特异性CTL活性显著高于其他各组(P<0.05)。pFlt3L和pCCL5表达质粒联用可显著促进小鼠Th1型细胞因子的表达,并对带HBc抗原的DNA疫苗的免疫应答具有促进作用。
To observe the eukaryotic recombinant plasmid pFlt3L and pCCL5 carrying HBc antigen DNA vaccine-induced antigen-specific immune response. The pFlt3L and pCCL5 were transfected into Hep G2 cells by lipofectamine respectively. Then the biological activity of two cytokines, Flt3L and CCL5, in supernatants of the cells was detected by proliferation and chemotaxis chamber assays. The recombinant plasmid pFlt3L and pCCL5 alone or in combination with the DNA vaccine carrying HBc antigen were immunized by intramuscular injection. The proliferation of spleen lymphocytes was detected by MTT assay. The levels of IFN in spleen CD8 + T lymphocytes were detected by flow cytometry The expression of IL-4 in supernatant of spleen lymphocytes was detected by ELISA and the cytotoxic activity of CTL was detected by lactate dehydrogenase (LDH) release assay. Results: Bone marrow cell proliferation and chemotaxis experiments confirmed that both Flt3L and CCL5 have biological activity. pFlt3L and pCCL5 alone or in combination could promote specific lymphocyte proliferation (P <0.05), increase the expression of IFN-γ in CD8 + T lymphocytes (P <0.05 or P <0.01), IL-4 (P> 0.05). The specific CTL activity of splenocytes in Flt3L + CCL5 group was significantly higher than that in other groups (P <0.05). The combination of pFlt3L and pCCL5 expression plasmids could significantly promote the expression of Th1 cytokines in mice and promote the immune response of DNA vaccine with HBc antigen.