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目的 获得具有多种稀有酶位点和CMVIE增强子 启动子及SV40晚期poly(A)加尾信号的pSFV1载体系统。方法 首先将含有多种稀有酶位点的人工接头插入到pSFV1的BamHⅠ和SmaⅠ位点 (获得的载体称为mpSFV1) ,然后在mpSFV1和辅助载体Helper2的SpeⅠ和SphⅠ位点分别插入SV40晚期poly(A)加尾信号和CMVIE增强子 启动子序列。以间接免疫荧光法检测构建的表达载体mpSFV1 A CMV表达登革 2型病毒PrME基因的效率 ,并用RT PCR法鉴定辅助载体Helper2 A CMV包装形成重组病毒的能力。结果 经PCR和酶切鉴定证明 ,接头、CMVIE增强子 启动子序列和SV40晚期poly(A)加尾信号序列均已导入pSFV1载体系统中 ;测序结果也与已知序列一致。间接免疫荧光法证明 ,PrME蛋白在BHK2 1细胞中获得了表达 ,同时 ,改造后的辅助载体也具有包装形成重组病毒的能力。结论 已将以RNA为基础的pSFV1载体系统构建成以DNA为基础的表达载体系统。
Objective To obtain a pSFV1 vector system with a variety of rare enzyme sites and CMVIE enhancer promoter and SV40 late poly (A) tailed signal. Methods The artificial linker containing various rare enzyme sites was first inserted into the BamHI and SmaI sites of pSFV1 (the vector obtained was named mpSFV1), and then SV40 late poly (SV40) was inserted into SpeS and SphI sites of mpSFV1 and Helper2, respectively A) Tail signal and CMVIE enhancer promoter sequence. Indirect immunofluorescence was used to detect the efficiency of expression of the constructed expression vector mpSFV1 A CMV in Dengue 2 virus PrME gene and the ability of Helper2 A CMV packaging to form a recombinant virus was identified by RT-PCR. Results The PCR and restriction enzyme digestion proved that the promoter, CMVIE enhancer promoter sequence and SV40 late poly (A) tailed signal sequence were all introduced into pSFV1 vector system. The sequencing results were also consistent with the known sequences. Indirect immunofluorescence demonstrated that PrME protein was expressed in BHK21 cells and that the engineered helper vector also had the ability to package recombinant virus. Conclusion The RNA-based pSFV1 vector system has been constructed as a DNA-based expression vector system.