猪圆环病毒2型ORF2/猪白介素2嵌合基因真核表达载体的构建、表达及在小鼠体内的免疫效果研究

摘要：为了研究猪圆环病毒2型（PCV2）核酸疫苗，本研究采用重叠延伸PCR（splicing by overlap extension-PCR , SOE-PCR）方法通过一基因柔性接头（linker）(G4S)3将PCV2的ORF2全长基因和猪白介素2的成熟肽基因（PoIL-2）构建成PCV2-linker-PoIL-2嵌合基因并克隆入pGEM-T Easy载体，对阳性重组质粒进行双酶切后回收嵌合基因亚克隆入pcDNA3.1(+)真核表达载体中；筛选阳性重组表达质粒（rpcDNA3.1/PCV2-linker-PoIL-2）瞬时转染COS-7细胞，分别采用PCV2抗原间接免疫荧光试验和PoIL-2蛋白ELISA试验方法检测COS-7细胞中表达的重组融合蛋白(rCap-linker-PoIL-2 )生物学活性；提取rpcDNA3.1/PCV2-linker-PoIL-2免疫Balb/c小鼠，采用PCV2抗体ELISA检测试剂盒检测rpcDNA3.1/PCV2-linker-PoIL-2在小鼠体内的免疫效果，并与只含PCV2 ORF2基因的重组表达质粒pcDNA3.1/OFR2 (rpcDNA3.1/OFR2)进行免疫效果比较。结果表明：成功构建了PCV2-linker-PoIL-2嵌合基因及其pcDNA3.1(+)重组表达质粒；rpcDNA3.1/PCV2-linker-PoIL-2在COS-7细胞内进行了成功表达，表达的rCap-linker-PoIL-2蛋白存在于COS-7细胞的细胞浆内，可与抗PCV2和抗PoIL-2蛋白抗血清发生特异性免疫反应，表明rCap-linker-PoIL-2蛋白具有Cap蛋白和PoIL-2蛋白的双重生物学活性； rpcDNA3.1/PCV2-linker-PoIL-2质粒免疫小鼠后可诱导小鼠产生明显的抗PCV2抗体，且其诱导的抗体水平明显高于rpcDNA3.1/OFR2。本研究为PCV2的核酸疫苗研制奠定了基础。

Study on the Construction, Expression and Immune Effect in mice of the Eukaryotic Expression Vector of Porcine Circovirus Type 2 ORF2/Porcine Interleukin-2 Chimeric Gene

Abstract: To exploit the porcine circovirus type 2 (PCV2) DNA vaccine to prevent the PCV2 disease, the recombination chimeric gene of PCV2-linker-PoIL-2 constructed by PCV2 ORF2 gene linked porcine interleukin-2 (PoIL-2) mature peptide gene via a 15-amino acid glycine-rich linker linker,（G4S）3] by SOE-PCR (splicing by overlap extension-PCR) method was cloned into pGEM-T Easy vector and subsequently sub-cloned into eukaryotic expression vector pcDNA3.1(+). The positive recombination expression plasmid of pcDNA3.1/PCV2-linker-PoIL-2 (rpcDNA3.1/PCV2-linker-PoIL-2) was selected and transfected into COS-7 cells by lipofectamine. The bioactivities of the expressed recombinant fusion protein (rCap-linker-PoIL-2 protein) in COS-7 cells were tested by the methods of indirect immunofluorescent assay (IFA) for detection PCV2 antigen and porcine IL-2 ELISA assay for detection PoIL-2 protein, respectively. The immune effects in Balb/c mice induced by rpcDNA3.1/PCV2-linker-PoIL-2 and recombinant expression plasmid of pcDNA3.1/OFR2 (rpcDNA3.1/OFR2) were evaluated by ELISA assay for detection PCV2 antibody. The result showed that the chimeric gene of PCV2-linker-PoIL-2 and the rpcDNA3.1/PCV2-linker-PoIL-2 were successfully obtained and constructed, respectively. The rCap-linker-PoIL-2 protein, which was existed in the cytoplasm of COS-7 cells and expressed by rpcDNA3.1/PCV2-linker-PoIL-2, displayed the specific immune response for anti-PCV2 and anti-PoIL-2 antibody by IFA and ELISA assay, respectively, which indicated the rCap-linker-PoIL-2 protein had the duplex bio-activity of Cap protein and PoIL-2 protein. Although the specific antibody response to PCV2 in mice could be induced by being intramuscularly immunized with ether rpcDNA3.1/PCV2-linker-PoIL-2 or the rpcDNA3.1/OFR2, the antibody titer induced by former was much higher than that caused by the latter after the second and third immune. Our study laid the good foundation for further PCV2 DNA vaccine development.