SUSD2基因真核表达载体构建及其在H322细胞中表达  

Construction of eukaryotic expression vector of sushi domain containing 2 and it's expression in H322 cell

作　　者：蔡翠霞[1] 刘心蕊[1] 王涵多[1] 高媛[1] 王飞[2] Cuixia Cai Xinrui Liu Handuo Wang Yuan Gao Fei Wang(Institute of Genetic Engineering, School of Basic Medical Science, Southern Medical University, Guangzhou 510515, China Higher Education Research Center, Southern Medical University, Guangzhou 510515, China)

机构地区：[1]南方医科大学基础医学院基因工程研究所,广东广州510515 [2]南方医科大学高等教育研究中心,广东广州510515

出　　处：《生物技术》2016年第5期421-425,共5页Biotechnology

摘　　要：[目的]构建SUSD2基因真核表达载体,并观察其在真核细胞H322细胞中的表达。[方法]从H322细胞中提取总RNA并逆转录为c DNA,采用PCR技术扩增出含有EcoRⅠ和HindⅢ酶切位点的人SUSD2基因片段,以质粒pc DNA3.1为表达载体,构建重组质粒pc DNA3.1-SUSD2。采用PCR、双酶切鉴定以及测序验证c DNA片段大小和序列正确。将重组载体pc DNA3.1-SUSD2转染H322细胞,Western Blot法检测SUSD2蛋白的表达。[结果]PCR、双酶切鉴定以及测序结果显示pc DNA3.1-SUSD2包含大小、序列正确的SUSD2片段;Western Blot结果显示SUSD2蛋白在转染pc DNA3.1-SUSD2的H322细胞中表达高于转染pc DNA3.1的H322细胞。[结论]成功获得SUSD2基因全长序列,并成功构建SUSD2基因真核表达载体,有效地在非小细胞肺癌细胞株H322中表达,为进一步研究该基因功能及机制奠定了基础。[ Objective ] To construct eukaryotic expression vector of sushi domain containing 2 （SUSD2） gene, and to examine its expression in H322 cell. [ Methods ] Total RNA was extracted from the H322 cells and reversely transcribed. The full length SUSD2 cDNA containing two restriction sites （EcoR Ⅰ and Hind Ⅲ） was synthesized by PCR. Recombinant plasmid pcDNA 3.1 - SUSD2 was constructed Basing on plasmid pcDNA3.1 expression vector. The size and sequence of cDNA fragments were confirmed by PCR and Hind Ⅲ and EcoRⅠ double digestion and sequencing. H322 cells were transfected with the pcDNA3.1 -SUSD2 vectors respectively and then the expression of SUSD2 protein was detected by Western Bloting. [ Results] The pcDNA3.1 - SUSD2 contained SUSD2 fragments of proper size and sequence by the results of PCR, double digestion and sequencing. And significantly increased SUSD2 protein expression in the H322 cells was detected by Western bloting after transfection 48 h. [ Conclusion ] pcDNA3.1 - SUSD2 was successfully constructed and expressed effectively in H322 ceils, which laid a foundation of further research on function and mechanism of SUSD2.

关 键 词：SUSD2 真核表达载体 人非小细胞肺癌细胞株H322 

分 类 号：Q782[生物学—分子生物学]