| 陆丽峰，苏波，姜浩，戴文香，向姝霖，唐海林，凌晖，苏琦.Chk1/2转基因MGC803细胞系的建立与鉴定.[J].中南医学科学杂志.,2013,41(2):140-145. |
| Chk1/2转基因MGC803细胞系的建立与鉴定 |
| Construction and Identification of Transfection of Human Chk1/2Gene in Gastric Cancer MGC803 Cells |
| 投稿时间：2012-04-05 |
| DOI： |
| 中文关键词: Chk基因 真核表达 转染 人胃癌MGC803细胞 |
| 英文关键词:Chk1/2 gene eukaryotic expression transfection human gastric cancer MGC803 cells |
| 基金项目:国家自然科学基金（30600285，81102854），湖南省自然科学基金重点项目(07JJ3033)，湖南省教育厅科学研究重点项目(09A077) |
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| 中文摘要: |
| 目的构建Chk1/2基因的真核表达载体和建立高表达Chk1/2基因人胃癌MGC803细胞，为研究Chk1/2功能奠定基础。方法参照野生型Chk1/2基因mRNA全序列，在cDNA两端各设计一条对应引物，并引入各自的酶切位点。从人胃癌MGC803细胞中提取mRNA作为模板合成Chk1/2 cDNA第一链，并扩增目的基因全表达序列片断，双酶切后定向克隆至pcDNA3.1(+)真核表达载体，经氨苄青霉素筛选阳性重组质粒，菌液PCR及测序法对重组质粒进行鉴定。用脂质体将重组质粒转染入MGC803细胞，经G418筛选后，RT-PCR及Western blot检测表达产物。结果菌液特异性PCR结果表明克隆的基因片断分别为1.4 kb和1.6 kb，测序分别证实为Chk1和Chk2基因，经NCBIBLAST分析与Genbank中Chk1基因的同源性为100%，编码的氨基酸同源性为100%，Chk2基因的同源性为99%，编码的氨基酸同源性为100%。G418筛选转染细胞4周后获得稳定转染空载体pcDNA3.1(+)的细胞克隆株和稳定转染重组质粒的MGC803细胞。经RT-PCR及Western blot 鉴定，Chk1/2在MGC803细胞中的表达比对照组明显增加。结论成功构建Chk1/2基因真核表达载体与Chk1/2高表达MGC803细胞，为研究二烯丙基二硫诱导MGC803细胞G2/M期阻滞的机制奠定了基础。 |
| 英文摘要: |
| ObjectiveTo construct the eukaryotic expression vector and human gastric cancer MGC803 cells of human Chk1/2 gene for investigating the function of Chk1/2.MethodsA pair of primer smatching each end of Chk1 and Chk2 cDNA and supplementing with appropriate endonuclease sites were designed according to the sequence published in NCBIGenBank.The Chk1 and Chk2 mRNA were extracted from human gastric cancer MGC803 cells,and used as the template to synthesize the first strand of cDNA.The Chk1 and Chk2 gene with complete expression sequence were amplified,which were cloned into pcDNA 3.1(+) vector after being double digested by endonucleases.The recombinants were identified with specific PCR and sequencing.The recombinant plasmid were transfected into MGC803 cells via lipofectamine,and after G418 selection,the expression of Chk1/2 were detected by RT-PCR and Western blot.ResultsThe results of specific PCR showed that a 1.4 kb and 1.6kb fragment had been cloned into pcDNA3.1(+) vector,which was further identified by sequencing and NCB BLAST analysis.The stable clones transfected with 2 vectors were respectively constructed.As identified by RT-PCR and Western blot,the expression of Chk1/2 in MGC803 cells were significantly increased as compared with that in control groups.ConclusionEukaryotic expression plasmid pcDNA3.1(+)/Chk1/2 and MGC803 cells of overexpression Chk1/2 have been constructed successfully,which may facilitate further research of Chk1 and Chk2 in G2/M arrest induced by diallyl disulfide. |
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