亚洲Ⅰ型口蹄疫病毒VP1基因真核表达载体的构建及其免疫活性的初步分析

Construction of recombinant plasmid with VP1 gene against AsiaⅠ FMDV and elementary analysis of its immunological activity

提取亚洲Ⅰ型口蹄疫病毒总RNA,用特异性引物通过RTPCR反应获得了约750bp的核酸片段,将其与pGEMTEasy载体连接,并转化大肠埃希氏菌JM109。重组质粒经PCR、酶切鉴定及测序,证明所克隆的片段含有完整的VP1基因;将重组质粒与表达载体pcDNA3.1(+)分别用BamHⅠ和EcoRⅠ双酶切后连接,构建成重组表达质粒,转化宿主菌DH5α,筛选阳性克隆。测序结果证明,目的基因正确插入到表达载体中,命名为pcDNAV;用同样方法将IL18基因插入pcDNAV中,命名为pcDNAVI。用pcDNAVI免疫豚鼠,2周后加强免疫1次,每周用ELISA检测豚鼠血清中的抗体水平。结果表明,pcDNAVI可引发机体产生体液免疫。

Total RNA was extracted from AsiaⅠ type of FMDV and the cDNA fragment of VP1 was amplified with specific primers by RT-PCR. The purified cDNA fragment was inserted into pGEM-T Easy vector. The recombinant plasmid was identified by restriction endonuclease analysis and PCR.It was proved by DNA sequencing that the acquired recombinant contains complete VP1 gene.Afterwards the complete VP1 gene from the identified recombinant was amplified with another pair of primers containing BamHⅠ and EcoRⅠ sites.The expression vector pcDNA3.1(+) was digested by BamHⅠ and EcoRⅠ respectively. The positive clone pcDNAV was identified. After IL-18 gene was inserted into pcDNAV by the same method, the eukaryotic expression recombinant plasmid pcDNAVI was constructed successfully. Guinea pigs were immunized with pcDNAVI plasmid,and a boost vaccination was given two weeks later.Kinetic tests were carried out by ELISA. The results showed that humoral immunoresponse were elicited by the pcDNAVI DNA vaccine.