CRT/MAGE-A3重组腺病毒载体的构建及体外表达的研究  

Construction and Expression of Recombinant Adenovirus for CRT/MAGE-A3

作　　者：刘新莉[1] 陈洋[1] 马萍[1] 

机构地区：[1]中国医科大学附属第一医院肿瘤研究所,沈阳110001

出　　处：《中国医科大学学报》2009年第7期493-496,共4页Journal of China Medical University

基　　金：辽宁省自然科学基金资助项目(20042073)

摘　　要：目的构建钙网织蛋白/黑素瘤抗原基因-A3(CRT/MAGE-A3)双基因重组腺病毒载体,并进行体外表达研究。方法从表达质粒pcDNA3/CRT中切取目的片段CRT,定向克隆至穿梭载体pShuttle-GFP-CMV。根据Genbank中提供的黑素瘤基因序列,设计并合成引物,以人肺癌组织cDNA文库为模板采用RT-PCR技术扩增人黑素瘤抗原基因-A3基因片段,测序后将黑素瘤抗原基因-A3基因片段定向克隆至穿梭载体,进而将穿梭载体克隆至PacⅠ限制性内切酶线性化的腺病毒载体pAdxsi。结果目的片段CRT转入穿梭载体后,酶切鉴定pShuttle(-ΔGFP)-CRT正确。人肺癌组织经RT-PCR扩增出大小约950 bp的基因片段,测序证实为MAGE-A3 DNA后,克隆至穿梭载体pShuttle(-ΔGFP)-CRT构建穿梭载体pShuttle-CRT-MAGE-A3,酶切鉴定证实构建正确。酶切穿梭载体将双基因片段克隆至腺病毒载体pAdxsi得到Ad-CRT/MAGE-A3病毒载体,酶切鉴定证实构建成功。转染293LP细胞并用Western blot检测到其体外表达。结论成功构建CRT/MAGE-A3重组腺病毒载体并证实其能在体外有效表达CRT蛋白及MAGE-A3蛋白。Objective To construct a recombinant replication-deficient adenovirus for calreticulin/melanoma antigen gene-A3 and assay its expression in vitro. Methods Cut the fragment of CRT from the expression plasmid pcDNA3/CRT, then cloned the gene fragment into the shuttle plasmid pShuttle-GFP-CMV. The cDNA for MAGE-A3 was got by Touch Down RT-PCR amplification,then it was confirmed by direct sequencing. Subsequently, subclone the fragment into the shuttle plasmid. Further, the aimed fragments were cloned into the Pac Ⅰ restriction enzyme linearizationed adenoviral vector pAdxsi. The resulting recombinant adenoviral vector was confirmed by PCR and Western blot. Results MAGE-A3 was correct in sequence. The recombinant plasmids were digested and identified. Western blot analysis showed the expression of CRT and MAGE-A3 in vitro. Conclusion The CRT/MAGE-A3 recombinant adenovims vector was successfully constructed. And its expression in vitro was confirmed.

关 键 词：钙网织蛋白 黑素瘤抗原基因-A3 腺病毒 基因治疗 

分 类 号：R73[医药卫生—肿瘤]