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pcD-NA3.1(+)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。
Objective To construct the eukaryotie expression vector and human gastric cancer MGC803 cells of hu- man Chkl/2 gene for investigating the function of Chkl/2. Methods A pair of primer smatching each end of Chkl and Chk2 eDNA and supplementing with appropriate endonuelease sites were designed according to the sequence published in NCBIGenBank. The Chkl and Chk2 mRNA were extracted from human gastric cancer MGC803 cells,and used as the template to synthesize the first strand of eDNA. The Chkl and Chk2 gene with complete expression sequence were amplified, which were eloned into peDNA 3.1 （ ＋ ） vector after being double digested by endonueleases. The recombinants were identified with specific PCR and sequencing. The recombinant plasmid were transfected into MGC803 cells via lipofectamine,and after C,418 selection,the expression of Chkl/2 were deteeted by RT-PCR and Western blot. Results The 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 transfeeted 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. Conclusion Eukaryotic expression plasmid pcDNA3. 1 （ ＋ ）/Chkl/2 and MGC803 cells of overexpression Chk1/2 have been constructed successfully,which may facilitate further research of Chkl and Chk2 in GJM arrest induced by diallyl disulfide.
出处
《中南医学科学杂志》
CAS
2013年第2期140-145,共6页
Medical Science Journal of Central South China
基金
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)
关键词
Chk基因
真核表达
转染
人胃癌MGC803细胞
Chk1/2 gene
eukaryotic expression
transfection
human gastric cancer MGC803 ceils
