【作者】 张志武；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 硕士

【摘要】 目的:构建CD80-pIRES-SART3真核表达载体。方法:首先用RT-PCR技术扩增出小鼠的CD80基因片段全长并在其两端加上酶切位点EcoR I,琼脂糖凝胶电泳并进行胶回收,纯化扩增出来的CD80目的基因片段,用同源重组技术将其插入线性化的真核表达载体pIRES的多克隆位点的EcoR I酶切位点中,转化感受态细胞,挑选阳性克隆株并大量扩增,提取阳性克隆株的质粒,通过PCR技术、双酶切以及测序检测CD80基因的插入情况;然后以PCR技术扩增出SART3全长基因片段并在其两端加上酶切位点Xba I,琼脂糖凝胶电泳并进行胶回收,纯化扩增出来的SART3目的基因片段,用同源重组技术将SART3基因插入真核表达载体CD80-pIRES上的Xba I酶切位点中,转化感受态细胞,挑选阳性克隆株并大量扩增,提取阳性克隆株的质粒,通过PCR技术、双酶切以及测序检测SART3基因的插入情况。结果: 1.成功提取小鼠脾脏的RNA并逆转录成cDNA,利用合成的引物扩增出小鼠CD80基因序列全长。2.成功将CD80基因片段插入pIRES真核表达载体多克隆位点的EcoR I酶切位点中,构建出CD80-pIRES真核表达载体。3.利用合成的引物成功扩增出SART3基因序列全长。4.成功将SART3基因片段插入CD80-pIRES真核表达载体中的Xba I酶切位点上,构建出CD80-pIRES-SART3真核表达载体。结论:成功构建CD80-pIRES-SART3真核表达载体,为进一步研究其在骨肉瘤基因免疫治疗中的作用奠定了基础,为进一步研究其在相关肿瘤基因免疫治疗中的作用奠定了基础,为肿瘤基因免疫治疗提供了可供参考的方法。更多还原

【Abstract】 Objective: Construction of eukaryotic expression vector of CD80-pIRES-SART3.Methods: Firstly,by using RT-PCR technolgy amplify the whole fragment of CD80 gene of mouse and to add EcoR I restriction enzyme cutting site on the both ends of the gene fragment.Then purify the purpose extract of CD80 fragment after agarose gel electrophoresis and gel extraction.Next,insert the CD80 gene fragment into EcoR I restriction enzyme cutting site of the linearized eukaryotic expression vector of pIRES by homelogens DNA technology. Afterward, get the recombinant vector into competence cells and choose positive cloning strains. Aμgment the positive cloning strains and extract the plasmid from the strains. Detect the recombinant vector by PCR technology、enzyme digest and gene sequence analysis. Meanwhile,by using PCR technolgy amplify the whole fragment of SART3 gene and to add Xba I restriction enzyme cutting site on the both ends of the gene fragment. Then purify the purpose extract of SART3 fragment after agarose gel electrophoresis and gel extraction. Next,insert the SART3 gene fragment into Xba I restriction enzyme cutting site of the linearized eukaryotic expression vector of CD80-pIRES by homelogens DNA technology.Finally, get the recombinant vector into competence cells and choose positive cloning strains. Aμgment the positive cloning strains and extract the plasmid from the strains. Detect the recombinant vector by PCR technology、enzyme digest and gene sequence analysis.ResμLts: 1.SuccessfμLly extract the RNA of mouse’s spleen and reverse transcribe the RNA into cDNA.Using synthetized primer amplify the whole fragment of CD80 gene. 2. SuccessfμLly insert the CD80 gene fragment into eukaryotic expression vector of pIRES and construct the CD80-pIRES vector.3. By using PCR technolgy amplify the whole fragment of SART3 gene.4. SuccessfμLly insert the SART3 gene fragment into eukaryotic expression vector of CD80- pIRES and construct the CD80-pIRES-SART3 vector.Conclusion: SuccessfμLly construct the CD80-pIRES-SART3 vector.This will lay a foundation for further study of gene immunotherapy of osteosarcoma,also lay a foundation for further study of gene immunotherapy of interrelated tumour and provide a referable strategy for gene immunotherapy of tumour.更多还原