摘要
目的通过基因工程技术构建人α1,2岩藻糖苷转移酶(ht)基因表达载体,以期HT在猪细胞表达,削减异种抗原α-Gal的合成。方法HindⅢ/XbaⅠ双酶切pcDNA3和pRc/CMV-htcDNA2种质粒,回收所需酶切片段,进而连接、转化感受态细菌,纯化pcDNA3-htcDNA重组质粒,对其进行多酶切、PCR和测序鉴定。结果多酶切反应产物电泳可见内切酶PvuⅠ酶切产生6.5kb片段,BglⅡ酶切产生4.6kb和1.9kb片段,ApaⅠ酶切产生5.6kb和0.9kb片段,HindⅢ/XbaⅠ酶切产生5.4kb和1.1kb片段,结果与设计相符。PCR反应扩增出1098bp的htcDNA核心片段。序列测定结果与Genbank中htcDNA序列比对,表明在pcDNA3质粒多克隆位点成功定向连接了htcDNA全长序列。结论成功构建了pcDNA3-htcDNA重组质粒。
Objective To construct the recombinant plasmid pcDNA3-htcDNA for expressing human α 1,2-fucosyltransferase （Ⅰ-Ⅱ） in pig cells by gene transfection in order to reduce the production of xenoantigen α-Gal. Methods The pcDNA3 and pRc/CMV-htcDNA were digested by Hind m and Xba Ⅰ ,then aimed fragments were recovered and linked. The recombinant plasmid pcDNA3-htcDNA was identified using muhidigestion, PCR and sequence analysis. Results pcDNA3-htcDNA was digested correctly into 6.5 kb,4.6 kb/1.9 kb,5.6 kb/0.9 kb and 5.4 kb/1.1 kb by Pvu Ⅰ ,Bgl Ⅱ ,Apa Ⅰ and Hind m/Xba Ⅰ respectively. The htcDNA 1 098 bp core fragment was amplified by PCR. Comparing to the sequence of Genbank it was identified that htcDNA total sequence was cloned into pcDNA3. Conclusion pcDNA3-htcDNA recombinant plasmid is successfully constructed in this study.
出处
《生物医学工程与临床》
CAS
2006年第2期105-108,共4页
Biomedical Engineering and Clinical Medicine
基金
天津市科委科技发展计划重点资助项目(编号:043803411)
