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

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合终止密码子的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clL-2基因真核分泌型表达载体，pcDNA-cIL-2和pcDNA-cIL-2／MH。将pcDNA-oIL-2／MH表达栽体通过磷酸钙方法转染HEK293T细胞进行瞬时表达，以确定构建的表达栽体能否介导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栽体作为阴性对照）。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1：5120，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7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CPVVP2基因疫苗的免疫应答水平。

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

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 levels in the medium of lymphocytes isolated from co-immunized mice was sig- nificantly higher than that of the lymphocytes from VP2-immunized and the negative control mice （P〈0.01）. In conclusion, IL-2 gene expression vector can significantly improve the CPV VP2 DNA vaccine immunogenecity.