中文摘要：

【目的】通过融合基因表达载体和共免疫基因表达载体研究大肠杆菌不耐热肠毒素（LT）B亚基基因对犬细小病毒VP2DNA疫苗免疫应答的影响。【方法】提取大肠杆菌44815菌株基因组DNA,通过PCR方法从基因组DNA中扩增LTB基因,同时采用PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2的主要抗原表位基因（VP2-70,编码70个氨基酸）。将上述基因分别连接到含有人CD5信号肽序列的载体pcDNA-CD5sp上,分别构建成它们的分泌型真核表达载体,pcDNA-CD5sp-LTB和pcDNA-CD5sp-VP2-70。再利用酶切连接的方法构建LTB与VP2-70融合的真核表达载体pcDNACD5sp-LTB-VP2-70。然后用pcDNACD5sp-VP2-70（VP2-70组）、pcDNACD5sp-LTB-VP2-70（VP2-LTB融合组）、pcDNA-CD5sp-LTB/pcDNACD5sp-VP2-70（VP2-LTB共免疫组）和pcDNA3.1A（空载体对照组）分别免疫小鼠。免疫后用间接ELISA检测不同时间小鼠血清的抗体水平,用MTT方法检测小鼠免疫5周后脾脏淋巴细胞的增殖活性。【结果】经过测序表明本研究扩增的LTB和VP2基因序列和构建的相关表达载体结构正确。通过Western-blot检测证明构建的表达载体均能介导相应基因在真核细胞进行分泌表达。ELISA检测结果表明,3组实验组小鼠接受VP2DNA疫苗免疫后均能产生特异的体液免疫应答反应,特别是VP2-LTB基因融合组小鼠的抗体水平在第5周时高达1：5120,明显高于其它两组（P〈0.01）。3组免疫小鼠抗体的亚型均表现IgG1抗体水平明显高于IgG2a抗体水平（P〈0.01）。淋巴细胞增殖实验结果表明,在ConA的刺激下,3组免疫小鼠的淋巴细胞刺激指数均明显高于对照组（P〈0.01）,说明VP2DNA疫苗能够引起淋巴细胞的增殖。但3组免疫小鼠之间的刺激指数没有明显差异（P〉0.05）。【结论】在小鼠体内,LTB基因表达载体可明显提高CPVVP2DNA疫苗的体液免疫应答水平。

英文摘要：

[Objective]To investigate the effect of Escherichia coli heat-labile enterotoxin （LT） B subunit （LTB） gene on canine parvovirus （CPV） VP2 gene vaccine.[Methods]The LTB gene was amplified by PCR from genomic DNA of E.coli 44815 strain.The VP2-70 fragment （210 bp） encoding major epitope of VP2 （70 amino acids） was amplified by PCR from a plasmid encoding VP2 gene.VP2-70 and LTB genes were inserted into the eukaryotic vector to construct VP2-70 gene,LTB gene and VP2-70-LTB fused gene vectors.The mice were immunized with VP2-70 vector,VP2-70-LTB fused vector,or VP2-70 vector plus LTB vector,respectively.The antibody titers at the different time were measured by using ELISA method.The spleen lymphocyte proliferation activity was analyzed by 3-（4,5-Dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide （MTT） assay.[Results ]The sequence of VP2-70 and LTB genes was identified.The recombinant VP2-70 and LTB proteins could be expressed in HEK293T cells in a secretory manner.The mice immunized with VP2-70 vector,VP2-70-LTB vector or VP2-70 vector plus LTB vector could generate the specific antibody against VP2 protein.The antibody titer immunized with VP2-70-LTB vector reached 1：5120 at 35 d post immunization,significantly higher than that of other two groups （P 0.01）.For antibody isotype analysis,the IgG1 isotype antibody titers in all test groups were significantly higher than of IgG2a （P 0.01）.The high-level spleen lymphocyte stimulation index was observed in the three test groups under the stimulation with Con A,higher than that in control groups （P 0.01）.[Conclusion]LTB gene could enhance the humoral immune response of CPV VP2 gene vaccine in mice.