犬GM-CSF基因对犬细小病毒VP2DNA疫苗的免疫增强作用  

Canine GM-CSF gene enhances the immunogenic potency of canine parvovirus VP2 DNA vaccine

作　　者：贾启恒[1] 温洁霞[1] 王利月[1] 林洪羽[2] 张峰[1] 王璐[1] 孙岩[1] 李秀锦[2] 仲飞[1] 

机构地区：[1]河北农业大学动物医学院/农业部动物疫病病原生物学华北科学观测实验站/河北省兽用生物制品工程技术研究中心,河北保定071001 [2]燕山大学环境与化学工程学院,河北秦皇岛066004

出　　处：《中国兽医学报》2013年第5期664-669,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30771586);河北省自然科学基金资助项目(C2008000244)

摘　　要：犬细小病毒编码的VP2蛋白是该病毒重要的结构蛋白和抗原蛋白。利用VP2基因制备的DNA疫苗能够刺激机体产生免疫应答反应。为进一步提高VP2DNA疫苗的免疫活性,本实验利用犬粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因作为生物佐剂研究其对犬细小病毒VP2DNA疫苗的免疫增强作用。首先通过RT-PCR方法从犬淋巴细胞中扩增GM-CSF基因,并将其插入到pcDNA3.1载体上,分别构建该基因的两个分泌型真核表达载体,即非融合表达载体pcDNA-cGMCSF和与Myc-His融合的表达载体pcDNA-cGMCSF/MH。用pcDNA-cGMCSF/MH载体转染HEK293T细胞以确定GM-CSF基因能否在真核细胞中进行分泌表达。然后用本室构建的VP2基因表达载体单免疫小鼠,用VP2表达载体与pcDNA-cGMCSF共免疫小鼠(pcDNA3.1空载体作为阴性对照)。免疫后用ELISA方法检测不同时间小鼠血清的抗体水平。用MTT法检测小鼠免疫后35d时淋巴细胞的增殖活性,同时用ELISA试剂盒检测小鼠淋巴细胞γ干扰素的表达水平。结果表明,本试验构建的表达载体能够介导重组GM-CSF在真核细胞中进行分泌表达。免疫实验表明,利用GM-CSF基因与VP2基因共免疫小鼠,抗体的水平明显高于VP2基因单免疫组(P<0.01)。共免疫组小鼠淋巴细胞的刺激指数和γ干扰素的表达水平均明显高于单免疫组(P<0.05)。由此可见,GM-CSF表达载体可明显提高CPV VP2DNA疫苗的免疫应答水平。VP2 protein, encoded by canine parvovirus （CPV） ,is a major structural protein and antigenic protein. The DNA vaccine derived from the VP2 gene can stimulate the immune responses to CPV. To enhance the VP2 gene vaccine potency, the dog granulocyte and macrophage colony- stimulating factor （GM-CSF） gene was used as a biological adjuvant to enhance VP2 gene vaccine potency. Firstly,GM-CSF gene was amplified by PCR and inserted into pcDNA3. 1A vector to construct two GM-CSF eukaryotic expression vectors：non-fusion vector pcDNA-cGM-CSF and Myc/His-fusion vector pcDNA-cGM-CSF/MH. Before immunization, the pcDNA-cGM-CSF/MH vector was transfected into HEK293T cells for transient expression to determine whether the recombinant cGM-CSF could be expressed in the eukaryotic cells in a secretory manner. The mice were separately immunized with VP2 vector （pcDNA-CDS-VP2,constructed previously in our laboratory） and VP2/cGM-CSF combined vectors （pcDNA3.1 vector as a negative control）. After immunization,the antibodies against CPV in the immunized mice were measured by ELISA, the spleen lymphocyte proliferation response was measured by lymphocyte proliferation assay, and the interferon-＇/expression activity of the mouse lymphocytes was measured by ELISA. The results showed that the recombinant cGM-CSF could be secretively expressed in HEK293T cells. Immunization results showed that the antibody level of the mice co-immunized with VP2 and cGM-CSF vectors was significantly higher than that immunized with VP2 vector alone （P〈0.01） at 35d after immunization. The lymphocyte stimulation index of VP2/cGM-CSF co-immunized mice was significantly higher than that of VP2 immunized mice （P〈0.05）. The IFN-γlevels in the medium of lymphocytes of the co-immunized mice was significantly higher than that of the VP2-immunized mice （P〈0.01）. In eonclusion,cGM-CSF gene can significantly improve the CPV VP2 DNA vaccine immunogenecity.

关 键 词：GM-CSF 免疫佐剂 VP2基因疫苗 犬细小病毒 

分 类 号：S852.65[农业科学—基础兽医学]