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目的构建婴儿双歧杆菌/胞嘧啶脱氨酶靶向性基因治疗系统。方法PCR扩增CD基因,EcoRⅠ,BamHⅠ对CD基因和pGEX-1LamdaT质粒同时进行双酶切,获得4.9kb、1.3kb两个DNA片段。T4DNA连接酶连接这两个片段构建重组的CD/pGEX一1LamdaT质粒,然后用电穿孔法将重组质粒转染婴儿双歧杆菌。从阳性转染的婴儿双歧杆菌中提取重组质粒,双酶切后检测切取片段长度,采用Sanger双脱氧链终止法对提取的重组质粒中的插入片段进行测序。结果从阳性转染的婴儿双歧杆菌中获得了6.2kb大小的重组质粒,该质粒经双酶切后,得到了4.9kb和1.3kb两个长度的片段,其长度分别与pGEX-1LambdaT及CD基因的长度相同。测序结果显示,提取的重组质粒中插入的基因片段全长及核苷酸序列与CD基因完全相同。结论外源性CD基因被准确插入pGEX-1LambdaT质粒并转入婴儿双歧杆菌中,婴儿双歧杆菌/胞嘧啶脱氨酶靶向性基因治疗系统被成功构建。
Objective To construct a Bifidobacterium infantis / cytosine deaminase targeted gene therapy system. Methods The CD gene was amplified by PCR. EcoRⅠ and BamHⅠ were double digested with CD gene and pGEX-1LamdaT plasmid respectively to obtain two DNA fragments of 4.9kb and 1.3kb. T4 DNA ligase ligated the two fragments to construct a recombinant CD / pGEX-1LamdaT plasmid, and then the recombinant plasmid was transfected into Bifidobacterium infantis by electroporation. The recombinant plasmids were extracted from the positive Bifidobacterium infantis. The length of the fragments was detected by double enzyme digestion and the inserted fragments in the extracted recombinant plasmids were sequenced by Sanger dideoxy chain termination method. Results A 6.2kb recombinant plasmid was obtained from the positive Bifidobacterium infants. The double digested DNA fragments of 4.9kb and 1.3kb were obtained. The lengths of the two fragments were the same as that of pGEX-1LambdaT and CD Genes of the same length. Sequencing results showed that the full length and nucleotide sequence of the inserted gene fragments in the recombinant plasmid were completely identical to the CD gene. Conclusion Exogenous CD gene was inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium infantis. The Bifidobacterium infantis / CDase targeted gene therapy system was successfully constructed.