基于胃癌MG7-Ag模拟表位基因疫苗的构建

Construction of a DNA vaccine based on the MG7-Ag mimotope of gastric cancer

目的 构建以胃癌 MG7- Ag模拟表位为基础的DNA疫苗 .方法 将 H ind 酶切位点、MG7- Ag模拟表位和通用性辅助性 T细胞 (Th细胞 )表位编码序列的反向互补序列顺次设计于下游引物中 ,通过 2次聚合酶链式反应将 2种表位连接于一段载体基因 ,H ind 酶切位点位于载体片段与表位基因之间 .将目的基因插入 p UCm - T载体 ,酶切鉴定和序列测定证实后 ,利用 p UCm- T载体和 pc DNA3.1(+)载体共有的 Kpn 和 Xba 酶切位点 ,将目的基因亚克隆入真核表达载体 pc DNA3.1(+)载体 .酶切鉴定后 ,利用目的基因和pc DNA3.1(+)载体上的 H ind 酶切位点 ,H ind 酶切去除载体基因片段 ,得到胃癌 MG7- Ag模拟表位和通用性 Th细胞表位的真核表达载体 .结果 经序列测定证实 :PCR产物为载体基因、H ind 酶切位点、MG7- Ag模拟表位及通用性Th细胞表位的融合片段 ;最终得到的重组 pc DNA3.1(+)质粒含有正确编码以上 2种表位的基因片段 .结论 以胃癌MG7- Ag模拟表位为基础的 DNA疫苗构建成功 。

AIM To construct a DNA vaccine based on the MG7 Ag mimotope of gastric cancer. METHODS Hin dⅢ site and reverse complementary sequence of MG7 Ag mimotope and a universal T helper cell (Th) epitope were included in the primers in tandem. By polymerase chain reaction (PCR), gene encoding the epitopes was incorporated into a fragment of carrier gene with the interval of Hin dⅢ site. The PCR product was then inserted into the pUCm T vector, which then underwent endonuclease digestion and sequencing. Sequentially, PCR product was subcloned into the pcDNA3.1(+) plasmid from the pUCm T vector by digestion with Kpn Ⅰand Xba Ⅰrestriction endonucleases, whose sites exist in both the vectors. The recombinant pcDNA3.1(+) was digested with two endonucleases to verify successful subcloning. Then Hin dⅢ endonuclease was used to remove the carrier gene from the recombinant pcDNA3.1(+) vector. The final pcDNA3.1(+) vector was sequenced to ensure the existence of gene that properly encodes the MG7 Ag mimotope and the Th epitope. RESULTS By sequencing, it was confirmed that: the PCR product was exactly the fused gene fragment of carrier gene, Hin dⅢ site, MG7 Ag mimotope and the universal Th epitope; The final recombinant eukaryotic expression vector pcDNA3.1(+) included properly encoded MG7 Ag mimotope and The epitope. CONCLUSION The DNA vaccine based on the MG7 Ag mimotope can be successfully constructed, providing the premise for further investigation of its potency in the immunotherapy of gastric cancer.