猪圆环病毒2型ORF2重组腺病毒的构建与鉴定及其免疫效果研究

Construction and Identification of Recombinant Adenovirus Containing Cap Gene of Porcine Circovirus Type 2

为了研究含有猪圆环病毒2型(porcinecircovirus type 2,PCV2)的重组腺病毒作为基因工程疫苗的潜在应用价值,本试验根据GenBank中猪圆环病毒2型基因序列,设计了1对引物,用于猪圆环病毒2型ORF2基因的扩增。将目的基因T/A克隆后,亚克隆至腺病毒转移载体pShuttle-CMV,构建重组穿梭质粒pShuttle-CMV-ORF2。经PCR方法和限制性内切酶酶切法及测序证明该基因已成功连接后,重组腺病毒转移载体经PmeⅠ酶切线性化,在BJ5183细菌中与腺病毒骨架载体pAdEasy-1同源重组获得重组腺病毒质粒pAd-CMV-ORF2。经PCR方法和PacⅠ酶切方法及测序鉴定表明该重组腺病毒载体已构建成功。PacⅠ酶切线性化pAd-CMV-ORF2,脂质体法转染AD293细胞进行病毒的包装和扩增。PCR及RT-PCR、IFA、Westernblotting检测目的基因及其表达。结果表明,本试验成功构建了重组腺病毒pAd-CMV-ORF2。3次噬斑试验纯化重组腺病毒,测得其TCID50为10-8.75/0.1mL。将重组腺病毒接种SPF级雌性BALB/c小鼠,用ELISA抗体检测试剂盒检测特异性抗体水平。小鼠免疫试验测得其特异性抗体水平较高,为PCV2重组腺病毒基因工程疫苗的进一步研究打下基础。

In order to research the feasibility of recombinant adenovirus containing Cap gene of porcine circovirus type 2 as genetically engineering vaccine,we designed a pair of primers according to PCV2 sequence. The primers were used for the amplification of the porcine circovirus type 2 ORF2 gene. The target gene was inserted into vector pMD18-T and then subcloned to the adenovirus shuttle plasmid pShuttle-CMV to construct the recombinant shuttle plasmid pShuttle-CMV-ORF2. After the vector was confirmed correct by PCR,restriction endonuclease digestion and sequencing,lineared by PmeⅠenzyme digestion and transformed into E.coli BJ5183-AD-1 containing plasmid pAdeasy-1 to form the homogeneous recombinant adenovirus plasmid vector pAd-CMV-ORF2. It was confirmed correct by PCR,PacⅠ digestion and sequencing. The PacⅠ digestion of linearized pAd-CMV-ORF2 was transfected into AD293 cells using lipofectamineTM2000 for virus packaging and amplification. The target gene and its expression in virus were detected by PCR,RT-PCR,IFA and Western blotting. Three times plaque experiments purified the recombinant adenovirus. The TCID50 was 10^-8.75/0.1 mL. It was indicated that the recombinant virus carrying the gene encoding the cap protein was successively constructed,and inoculated SPF-class female BALB/c mice with the recombinant adenovirus,and then detected specific antibody level with the ELISA antibody test kit. In these ways,immunization experiments in mice had measured the high levels of specific antibodies,thus provided basis for the further study with genetic engineering of recombinant adenovirus for the PCV2 vaccine.