摘要
目的利用简并引物PCR法及重叠PCR法构建HBsAg(S),HSV-2gD模拟抗原表位P6及天然抗原表位NP6,IL-18真核表达载体,并对其表达能力进行鉴定,为后期疫苗的研制奠定基础。方法采用简并引物PCR法扩增IL-18-P6(包括P6-IL-18)片段及IL-18-NP6(包括NP6-IL-18);根据GenBank(FJ589066.1)公布的S基因设计引物,扩增获得含有重叠区的S片段(包括S1,S2,S3,S4);采用重叠PCR法扩增三基因融合片段IL-18-P6-S,IL-18-NP6-S,S-P6-IL-18和S-NP6-IL-18,经纯化回收后将其克隆到原核载体pMD 18-T simple vector中,测序正确的目的片段插入真核表达载体pcDNA3.1(-),构建重组质粒pcDNA3.1-IL-18-P6-S,pcDNA3.1-IL-18-NP6-S,pcDNA3.1-S-P6-IL-18和pcD-NA3.1-S-NP6-IL-18,采用间接免疫荧光法检测靶基因的表达情况。结果成功构建了含有双抗原的真核表达载体pcDNA3.1-IL-18-P6-S,pcDNA3.1-IL-18-NP6-S,pcDNA3.1-S-P6-IL-18和pcDNA3.1-S-NP6-IL-18。脂质体介导真核表达载体转染CHO细胞,间接免疫荧光法检测细胞浆中有黄绿色荧光。结论构建的真核表达载体pcDNA3.1-IL-18-P6-S,pcDNA3.1-IL-18-NP6-S,pcDNA3.1-S-P6-IL-18和pcDNA3.1-S-NP6-IL-18能在CHO细胞中有效表达。
Objective Degenerate primer PCR and overlapping PCR were used to construct IL-18 eukaryotic expression plasmids for natural epitope NP6 and epitope P6 derived from HBsAg（S） and HSV-2gD. Expression of plasmids was determined, laying the foundation for subsequent research. Methods Fragments of IL 18-P6 （including fragments of P6- IL 18）, and IL-18-NP6 （including NP6 IL-18） were subjected to degenerate primer PCR. Primers were designed in ac cordance with the S gene in GenBank （FJ589066.1）, and S fragments containing the overlapping zone （including S1, S2, S3, and S4） were obtained. Fragments of the 4 fusion genes IL-18-P6-S, IL-18-NP6-S, S-P6-IL-18, and S-NP6-IL-18 were amplified by overlapping PCR, purified, and inserted into a pMD-18-T simple vector. Correct sequence fragments were inserted into the eukaryotic expression vector pcDNA3.1 （-）, and the recombinant plasmids pcDNA3.1-IL-18-P6-S, pcDNA3.1-IL-18-NP6-S, pcDNA3.1-S-P6 IL-18, and pcDNA3. 1 S-NP6-IL 18 were obtained. The expression of these genes was analyzed with an indirect immunofluorescence assay. Results The eukaryotic expression vectors of pcD NA3.1-IL-18-P6-, pcDNA3.1 IL-18-NP6 S pcDNA3.1 S-P6-IL-18, and pcDNA3.1-S-NP6 IL-18 were successfully con- structed to contain both antigens. The eukaryotic expression vectors were transduced into CHO cells with liposomes, and yellow-green fluorescence was detected by indirect immunofluorescenee in cytoplasm. Conclusion Vectors of pcDNA3. 1 IL-18-P6-S, pcDNA3. 1-IL-18-NP6-S, peDNA3.1-S-P6 IL-18, and pcDNA3. 1-S-NP6-IL 18 were successfully ex pressed in CHO ceils.
出处
《中国病原生物学杂志》
CSCD
北大核心
2012年第6期401-405,共5页
Journal of Pathogen Biology
基金
山东省自然科学基金项目(No.ZR2009CQ003)
山东省卫生厅资助课题(No.2007HZ037
No.2011QW029)
