Chk1和Chk2高表达人胃癌BGC823细胞的建立与鉴定

Construct and identify gastric cancer BGC823 cells of overexpression of Chk1 and Chk2

细胞周期检测点Chkl和Chk2在参与G2/M期起着重要作用,本研究构建Chk1/2的真核表达载体和建立高表达Chk1/2基因人胃癌BGC823细胞,进一步证明Chk1/2高表达对BGC823细胞G2/M期的影响。根据NCBI Gen Bank Chk1/2基因全序列,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及测序对重组质粒进行鉴定。用脂质体将重组质粒转染BGC823细胞,经G418筛选后,RT-PCR及Western blot检测表达产物。结果显示,菌落特异性PCR表明克隆的基因片断分别为1.4 kb和1.6 kb,经测序与NCBIBLAST分析证实为Chk1和Chk2基因。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BGC823细胞,经RT-PCR及Western blot鉴定,Chk1/2在BGC823细胞中的表达较对照组与空载体组明显增加(P<0.05)。流式细胞术检测显示,Chk1转染组G2/M细胞较对照组与空载体组明显增加(P<0.05),而Chk2转染组G2/M细胞无明显差异(P>0.05)。上述结果表明,成功构建pcDNA3.1/Chk1与pcDNA3.1/Chk2真核表达载体和Chk1与Chk2高表达的BGC823细胞,Chk1高表达可阻滞BGC823细胞于G2/M。

Cell cycle checkpoint Chkl and Chk2 play an important role in the G2/M phase, this study constructs the eukaryotic expression vector of Chk1/2 and establishes human gastric cancer BGC823 cells of overexpression Chk1/2 gene, further proves the effect of G2/M phase in BGC823 cells of overexpression of Chk1/2. According to the whole sequence of NCBI GenBank chk1/2 gene, a corresponding primer was designed on each end of cDNA, and the respective enzyme cutting sites were introduced. mRNA was extracted from human gastric cancer cells as the first chain of a template synthesis of Chk1/2 cDNA, and fully expressed sequence amplification purpose gene fragments, after the double enzyme directional cloning to pcDNA3.1 eukaryotic expression vector, the ampicillin screening positive recombinant plasmid, microbial PCR and sequencing to identify recombinant plasmid. The recombinant plasmid was transfected into BGC823 cells with liposomes, and the expression of Chk1/2 were detected by RT-PCR and Western blot after G418 screening. The colony specific PCR showed that the cloned gene fragment was 1.4 KB and 1.6 KB respectively, which was confirmed by sequencing and NCBIBLAST analysis as Chk1 and Chk2 genes. The cell clones and stable transfection recombinant plasmid BGC823 cells were stable transfected with pcDNA3.1, and the expression of Chk1/2 in BGC823 cells was significantly increased by RT-PCR and Western blot （ P 〈0.05）. Flow cytometry detect showed that G2/M cells in overexpression of Chk1 obviously increased than in control and vector （ P 〈0.05）, and G2/M cells in overexpression of Chk2 had no difference in control and vector （ P 〉0.05）. The above results indicate that it is the successfully constructed pcDNA3.1/Chk1 and pcDNA3.1/Chk2 eukaryotic expression vectors and overexpression of Chk1 and Chk2 BGC823 cells that can block BGC823 cells in G2/M.