犬白细胞介素-7基因对犬细小病毒DNA疫苗的免疫增强作用

Immune Enhancing Effects of Canine Interleukin-7 Gene on Canine Parvovirus DNA Vaccine

【目的】白介素-7是动物体一种重要的多功能细胞因子,在促进B细胞、T细胞的形成和发育,在协同其它细胞因子提高机体免疫能力等方面发挥重要作用。本试验利用犬细小病毒VP2 DNA疫苗,在小鼠体内分析了犬白介素-7(cIL-7)基因的免疫增强作用。【方法】采用RT-PCR方法从犬脾淋巴细胞中扩增cIL-7基因,然后将cIL-7基因插入到真核表达载体pcDNA3.1A中,分别构建成与Myc/His标签融合和非融合的cIL-7真核分泌型表达载体pcDNA-cIL7/MH和pcDNA-cIL7。由磷酸钙介导将pcDNA-cIL7/MH质粒转染HEK 293T细胞使其进行瞬时表达,以Western-blot检测构建的表达载体能否介导cIL-7基因在真核细胞中进行分泌表达。用已构建的VP2表达载体pcDNA-CD5-VP2与pcDNA-cIL7载体共免疫小鼠,并设pcDNA-CD5-VP2单免疫小鼠和pcDNA-cIL7单免疫小鼠作为对照。免疫后通过ELISA方法检测抗体水平,并通过淋巴细胞增殖实验和ELISA方法分别检测免疫后35 d小鼠脾脏淋巴细胞刺激指数和γ-干扰素的表达水平。【结果】本试验扩增的cIL-7基因序列与GenBank中犬的序列完全一致。构建的表达载体能够介导cIL-7基因在HEK293T细胞中进行分泌表达。动物免疫试验结果显示,cIL-7与VP2共免疫组小鼠血清的抗体滴度和中和抗体效价分别极显著和显著高于VP2单免疫组(P<0.01和P<0.05);共免疫组小鼠淋巴细胞刺激指数和γ-干扰素表达水平也显著高于VP2单免疫组(P<0.05)。【结论】cIL-7基因可增强小鼠对VP2 DNA疫苗的免疫应答反应。

【Objective】 Animal interleukin-7（IL-7）,a multifunctional cytokine,plays an important role in B-and T-cell generation and development and in improvement of animal immune capacity coordinating with other cytokines.In this study,the immune enhancing effects of canine IL-7（cIL-7） gene was investigated in mice co-immunized with expression vectors of canine parvovirus VP2 DNA vaccines.【Method】Canine IL-7 genes,both with stop codon and without stop codon,were amplified from canine splenic lymphocytes by RT-PCR,and then inserted into the eukaryotic expression vector pcDNA3.1A to contruct the non-fused and Myc/His-tag-fused cIL-7 eukaryotic secretory expression vectors（pcDNA-cIL7 and pcDNA-cIL7/MH）,respectively.To determine whether the expression vector could mediate cIL-7 gene expression in eukaryotic cells,the pcDNA-cIL7/MH plasmid was transfected into HEK293T cells using calcium phosphate transfection method for transient expression.The mice were co-immunized with VP2 gene expression vector（pcDNA-CD5-VP2,constructed previously in authors’ laboratory） and cIL-7 gene expression vector pcDNA-cIL7,the mice immunized with either pcDNA-cIL7 or pcDNA-CD5-VP2 were used as the control.After immunization,the antibodies against CPV in the immunized mice at different times were measured by ELISA.The spleen lymphocyte proliferation response at 35 d post-immunization was determined by lymphocyte proliferation assay,and the interferon-γ expression level of the mouse lymphocytes was measured by ELISA.【Results】 The results showed that the sequence of cIL-7 gene amplified was consistent with that in GenBank.Western-blot showed that the recombinant cIL-7 could be expressed and secreted in HEK293T cells.Immunization results showed that the antibody levels and the neutralizing antibody titers in the serum of VP2/cIL-7-immunized mice were significantly higher than that of VP2-immunized mice（P〈0.01 and P〈0.05）,respectively.The lymphocyte stimulation indexes and secreted IFN-γ levels of the VP2/cIL-7-immunized mice were significantly higher than that of VP2-imunized mice（P〈0.05）,respectively.【Conclusion】The cIL-7 gene can significantly enhance the immune response of the mice to CPV VP2 DNA vaccine.