牛白细胞介素-4真核表达载体的构建及免疫佐剂效应研究  

Construction of Bovine Interleukin-4 Gene Eukaryotic Expression Vector pVAX1-IL-4 and Its Effect of Vaccinee Adjuvant

作　　者：宫玉玲[1] 王克栋[1] 孙建华[1] 熊浩[1] 石明[1] 冉多良[1] 

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《新疆农业大学学报》2013年第4期259-264,共6页Journal of Xinjiang Agricultural University

基　　金：新疆维吾尔自治区高技术研究项目(201010101)

摘　　要：应用RT-PCR技术从植物血凝素(PHA)活化的牛外周血淋巴细胞中扩增出牛白细胞介素4(IL-4)特异性目的片段,将其克隆到真核表达载体pVAX1,双酶切及测序鉴定正确后,将构建好的重组质粒pVAX1-IL-4应用脂质体LipofectamineTM2000转染至293T细胞,经RT-PCR检测IL-4可进行转录表达,且MTT检测其具有生物学活性,表明IL-4在转染细胞中可以成功表达。然后将构建好的pVAX1-IL-4作为基因佐剂来研究其对牛病毒性腹泻粘膜病毒(BVD)核酸疫苗pVAV1-E0的免疫增强作用。经ELISA和MTT检测结果表明,pVAX1-IL-4同pVAV1-E0联合免疫组小鼠的特异性抗体水平、淋巴细胞增殖水平与pVAV1-E0单疫苗免疫组相比差异显著(P<0.05)。证明重组质粒pVAX1-IL-4可作为佐剂与核酸疫苗共同作用来提高后者的免疫原性。Bovine interleukin-4 cDNA was synthesized by reverse transcription polymerase chain reaction （RT-PCR） from mRNA extracted of blood lymphocytes of which were stimulated by 10μg/mL PHA in vitro.Then Bovine interleukin-4 gene was inserted into eukaryotic expression vector pVAX1.The recombi- nant plasmid was identified by enzyme digestion.The recombinant plasmid was transfected by Lipofectami- neTM 2000 to 293T cells,expression of IL-4 gene was detected by RT-PCR and MTT,the results indicated that the BOIL-4 gene was successfully carried out and transiently expressed in 293T cells,and had some bi- ological activities.Then the recombinant plasmid pVAXI-IL-4 were used as an adjuvant to research the im- muno-enhancing effect on bovine viral diarrhea gene vaccine（pVAV1-E0）.And detected by using ELISA and MTT,the results showed the specific antibody level and proliferation of the tlymphocyte level of the mices immunized with both pVAXl-IL-4 and pVAV1-E0 were significantly higher than that of the mices immunized with pVAV1-E0 alone（ P 〈0.05）.That indicated the recombinant plasmid pVAXI-IL-4 can be used as an adjuvant to increase activity of the gene vaccine.

关 键 词：克隆 真核表达 白细胞介素-4 293T细胞 免疫佐剂 

分 类 号：S851.652.6[农业科学—预防兽医学]