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目的构建人乳头瘤病毒(HPV)6b型结构蛋白L1-黑色素瘤抗原(MAGE-A3)细胞毒T淋巴细胞(CTL)表位嵌合基因并表达目的蛋白,为下一步制备HPV6bL1/MAGE-A3-CTL嵌合疫苗提供理论基础和实验依据。方法将MAGE-A3 CTL表位基因插入到UPV6bL1的羧基端序列中,将目的基因连入真核表达转移载体pFastBac~(TM)1,构建重组质粒pFastBac~(TM)1/HPV6bL1/MAGE- A3-CTL,转化DH10Bac感受态细胞,获得重组杆状病毒Bacmid/HPV6bL1/MAGE-A3-CTL;将重组杆状病毒转染sf-9昆虫细胞表达嵌合目的蛋白,并经SDS-Page电泳、考马斯亮蓝染色及Western blot分析鉴定。结果成功构建了重组质粒pFastBac~(TM)1/HPV6bL1/MAGE-A3-CTL,获得了重组杆状病毒Bacmid/HPV6bL1/MAGE-A3-CTL,并在真核细胞sf-9昆虫细胞中成功表达了嵌合目的蛋白。结论通过基因克隆方法能成功的将MAGE-A3 CTL表位基因插入到HPV6bL1基因中,并能在真核细胞表达系统中表达出嵌合目的蛋白,为进一步制备HPV6bL1/MAGE-A3-CTL嵌合疫苗提供理论基础和实验依据。
Objective To construct the chimeric gene of cytotoxic T lymphocyte (CTL) epitope of human papillomavirus (HPV) 6b L1-melanoma antigen (MAGE-A3) and to express the target protein for further preparation of HPV6bL1 / MAGE-A3 -CTL chimeric vaccine to provide the theoretical basis and experimental basis. Methods The MAGE-A3 CTL epitope gene was inserted into the carboxyl terminal sequence of UPV6bL1. The target gene was inserted into the eukaryotic expression vector pFastBac ~ (TM) 1 to construct the recombinant plasmid pFastBac ~ (TM) 1 / HPV6bL1 / MAGE-A3 The recombinant baculovirus was transfected into sf-9 insect cells to express the chimeric protein. After SDS-PAGE electrophoresis, the recombinant baculovirus was transfected into DHF-9 cells to obtain recombinant baculovirus Bacmid / HPV6bL1 / MAGE-A3-CTL. Muscle blue staining and Western blot analysis. Results The recombinant baculovirus pFastBac (TM) 1 / HPV6bL1 / MAGE-A3-CTL was successfully constructed and the recombinant baculovirus Bacmid / HPV6bL1 / MAGE-A3-CTL was successfully expressed in eukaryotic sf-9 insect cells Chimeric protein of interest. Conclusion The MAGE-A3 CTL epitope gene can be successfully inserted into HPV6bL1 gene by gene cloning method, and the target protein can be expressed in eukaryotic expression system. To further prepare the HPV6bL1 / MAGE-A3-CTL chimera Vaccine provides the theoretical basis and experimental basis.