细胞因子GM-CSF对犬透明带3DNA疫苗免疫小鼠抗生育效果的研究

Effects of granulocyte-macrophage colony-stimulating factor （GM-CSF） on the anti-fertility efficacy of canine zona pellucida glycoprotein 3 （CZP3） DNA vaccine

目的粒细胞-臣噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating fac-tor，GM-CSF）作为免疫佐剂，能够影响犬透明带3（CZP3）DNA疫苗的免疫效果，有助于研制有效降低流浪犬生育的犬透明带3DNA疫苗。方法采用RT-PCR和实时荧光定量PCR（qRT-PCR）方法，分析GM-CSF对小鼠肌肉注射部位的抗原提呈细胞（antigenpresentingcells，APCs）成熟和富集的影响。然后通过电脉冲刺激小鼠肌肉，将分子佐剂pcDNA3-GM-CSF与DNA疫苗pcDNA3．CZP3单独或联合免疫接种雌性BALB／c小鼠。ELISA方法检测了小鼠血清和生殖道中抗体IgG和sIgA（secretaryIgA）的水平以及血清中IL4和IFN-1，的含量，MTT方法检Nd,鼠脾脏T细胞的增殖，间接免疫荧光验证血清中CZP3抗体与小鼠卵细胞结合的能力，抗生育实验分析免疫不育效果。结果GM-CSF与peDNA3．CZP3共免疫小鼠能够显著促进CD80、CD83和CD86的表达，促进pcDNA3-CZP3免疫接种小鼠血清中IgG和生殖道中sIgA抗体水平的显著升高（P〈0．0k）。免疫小鼠的IL-4和IFN-γ的含量均显著增加（P〈0．05），MTF法证明脾脏T细胞增殖显著（P〈O．05），间接免疫荧光结果表明小鼠卵细胞表面的荧光强度和致密度与免疫组血清中抗体的水平成正相关．抗生育实验表明GM-CSF显著性（P〈0．05）地降低DNA疫苗pcDNA3-CZP3免疫小鼠的产仔数目。结论GM-CSF作为免疫佐剂能够显著增强犬透明带3DNA疫苗的免疫不育效果，可以作为增强犬透明带3DNA免疫不孕疫苗的有效佐剂。

Objective To investigate the effects of GM-CSF （granulocyte-macrophage colony- stimulating factor） as an immune adjuvant on the anti-fertility efficacy of canine zona pellueida glycoprotein 3 （CZP3） DNA vaccine. Methods RT-PCR and quantitative real-time PCR were performed to analyze the effects of GM-CSF on the maturation of antigen presenting cells （APCs） and the enrichment of APCs at in- jection site. Female BALB/c mice were immunized with CZP3 DNA vaccine peDNA3-CZP3 alone or in com- bination with genetic adjuvant pcDNA3-GM-CSF by using the method of electrical impulses. ELISA （en- zyme-linked immunosorbent assay） was used to detect the levels of IgG and sIgA （ secretory IgA） antibodies in serum samples and genital tracts and the levels of IL-4 and IFN-γ in serum samples. MTT method was used to analyze the proliferation of spleen T cells in mice. The binding ability of serum anti-CZP3 antibody to native mouse oocytes was analyzed by indirect immunofluoreseence assay. All of the female BALB/c mice were coupled with male mice of the same age six weeks ＇after the first-dose vaccination. Litter size at birth in each group was counted and the differences between different groups were comparatively analyzed . Results Immunizing the mice with peDNA3-CZP3 and GM-CSF significantly promoted the expression of CD80, CD83 and CD86 （P〈0.05） and increased the sIgA antibody level in genital tract and IgG level in serum （P〈 O. 01 ）. Moreover, the levels of IL-4 and IFN-γ in serum samples were significantly up-regulated （P〈0.05） and the proliferation of spleen T cells was significantly enhanced （ P〈0.05 ）. Results of the indirect immu- nofluorescence assay showed that the fluorescence intensity and density on mouse egg surface were positively correlated with the level of antibody in serum. Results of the anti-fertility test suggested that GM-CSF signifi- cantly reduced the litter size in mice immunized with pcDNA3-CZP3 vaccine （ P〈O. 05 ）. Conclusion GM- CSF could be used as an effective adiuvant to enhance the anti-fertility efficacy of CZP3 DNA vaccine.