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目的克隆出针对表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)PEP-3表位的cDNA片段,为高表达EGFRvⅢ肿瘤的免疫治疗奠定基础。方法根据PEP-3的碱基序列设计出有12对互补碱基的正负引物,正负引物互为模板进行PCR扩增,制备出两端含酶切位点的PEP-3 cDNA片段,再将扩增的PCR产物亚克隆于载体pGEM-T Easy构建重组质粒pGEM-T Easy/PEP-3。结果经限制性内切酶酶切鉴定和DNA测序证实,编码PEP-3的cDNA片段被成功扩增并亚克隆于载体pGEM-T Easy中。结论成功克隆了编码表皮生长因子受体Ⅲ型突变体PEP-3表位的cDNA片段。
Objective To clone a cDNA fragment targeting the epidermal growth factor receptor type Ⅲ mutant (EGFRv Ⅲ) PEP-3 epitope and lay a foundation for the immunotherapy of EGFRv Ⅲ-overexpressing tumors. Methods According to the base sequence of PEP-3, 12 pairs of complementary primers were designed. The positive and negative primers were used as template to amplify the PEP-3 cDNA fragment. The amplified PCR product was subcloned into the vector pGEM-T Easy to construct the recombinant plasmid pGEM-T Easy / PEP-3. Results Restriction endonuclease digestion and DNA sequencing confirmed that the cDNA encoding PEP-3 was successfully amplified and subcloned into the vector pGEM-T Easy. Conclusion The cDNA fragment coding for epidermal growth factor receptor type Ⅲ mutant PEP-3 was successfully cloned.