白细胞介素-2基因表达质粒对犬细小病毒VP2DNA疫苗在小鼠的免疫增强作用  

Immunogenic enhancement of interleukin-2 expression plasmids for canine parvovirus VP2 DNA vaccine

作　　者：王璐[1] 李秀锦[2] 韩云珍[3] 王幸兴[1] 李振[1] 张峰[1] 潘红丽[1] 仲飞[1] 

机构地区：[1]河北农业大学动物科技学院,河北省兽医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 [3]福建农业职业技术学院,福建福州350119

出　　处：《中国兽医学报》2012年第11期1634-1639,1644,共7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蛋白是该病毒主要抗原蛋白。研究证实由VP2基因制备的DNA疫苗能够刺激机体产生免疫应答反应。为提高VP2DNA疫苗的免疫原性,本研究在小鼠体内尝试了利用犬白细胞介素2(cIL-2)基因增强VP2DNA疫苗免疫应答的研究。通过RT-PCR方法从犬脾淋巴细胞中分别扩增含终止密码子和不含终止密码子的cIL-2cDNA基因,然后将基因插入到真核表达载体pcDNA3.1中,分别构建成非融合的和与Myc/His融合的cIL-2基因真核分泌型表达载体,pcDNA-cIL-2和pcDNA-cIL-2/MH。将pcDNA-cIL-2/MH表达载体通过磷酸钙方法转染HEK 293T细胞进行瞬时表达,以确定构建的表达载体能否介导cIL-2在真核细胞中进行分泌表达。然后用VP2表达载体(pcDNA-CD5sp-VP2,本室构建)单注射和VP2/IL-2表达载体共注射对小鼠进行免疫(用pcD-NA3.1载体作为阴性对照)。免疫后通过ELISA方法检测免疫后不同时期小鼠血清VP2的抗体水平,并通过细胞增殖试验检测免疫后小鼠脾脏淋巴细胞的增殖反应,用ELISA方法测定小鼠淋巴细胞γ干扰素的表达水平。试验结果表明,扩增的小鼠cIL-2基因与GenBank的参考序列一致,构建的cIL-2表达载体能够介导重组cIL-2在HEK293T细胞中进行分泌表达。免疫结果显示,利用cIL-2/VP2表达载体共免疫小鼠,免疫后35d血清中VP2的抗体水平达到1∶5 120,明显高于VP2表达载体单免疫组(P<0.01)。淋巴细胞增殖试验表明,2组免疫小鼠的淋巴细胞刺激指数均明显高于阴性对照组(P<0.01),共免疫组的刺激指数又明显高于单免疫组(P<0.05)。共免疫小鼠淋巴细胞γ干扰素的表达水平明显高于单免疫组和阴性对照组(P<0.01)。由此可见,cIL-2表达载体可明显提高CPV VP2基因疫苗的免疫应答水平。Canine parvovirus （CPV） VP2 protein is the major antigenic protein. It was extensively used as a DNA vaccine to stimulate the immune responses against CPV. To improve the VP2 DNA vaccine poten- cy,we tested to use the canine interleukin-2 （cIL-2） gene as an adjuvant to enhance its immunogenicity. Briefly, the canine IL-2 cDNA fragments with nine lymphocytes by RT-PCR, and then the c pression vector to construct the non-fused and stop codon and without stop codon were amplified from ca- IL-2 genes were inserted into pcDNA3. 1A eukaryotic ex- the Myc/His-fused cIL-2 expression vectors,pcDNA-cIL-2and pcDNA-cIL-2/MH,respectively. To determine whether the expression vector can mediate cIL-2 secretory expression in eukaryotic cells, the pcDNA-cIL-2/MH plasmids were transfected into HEK293T cells mediate-d by calcium phosphate. The mice were separately immunized with VP2 vector （pcDNA-CDSsp-VP2 constructed previously in our laboratory） and VP2/cIL-2 combined vectors （pcDNA3.1 vector as a negative control）. After immunization,the serum levels of VP2 an tibodies were measured by ELISA,the spleen lymphocyte proliferation response was measured by lymphocyte proliferation assay,and the interferon-γ expression activity of the mouse lymphocytes was measured by ELISA. The results showed that the amplified eIL-2 gene sequence was identical with that published in GenBank, and the recombinant cIL-2 could be secretively expressed in HEK293T cells. Immunization results showed that the antibody level of the mice co-immunized with VP2 and cIL-2 vectors was up to 1 ： 5 120 at 35 d after immunization, significantly higher than that immunized with VP2 vector alone （P〈0.01）. Lymphocyte proliferation assay showed that the lymphocyte stimulation index of the two groups of VP2 gene-immunized mice were signif- icantly higher than that of the negative control group （P〈0. 01）, moreover, the index of VP2/eIL-2 co-immunized mice was significantly higher than that of VP2 immunized mice （P〈 0.05）. The IFN-γ lev

关 键 词：白细胞介素-2 生物佐剂 细小病毒 VP2 DNA疫苗 

分 类 号：S852.655[农业科学—基础兽医学]