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目的:检测人胃癌细胞系中FHIT基因mRNA的表达状况及构建pcDNA3.1-FHIT真核表达载体。方法:RT-PCR法检测三种不同类型人胃癌细胞系中FHIT基因mRNA的表达,构建真核表达质粒pcDNA3.1-FHIT,通过酶切法、PCR扩增法和DNA序列分析鉴定重组质粒后,用脂质体转染至FHIT基因mRNA阴性表达人胃癌细胞系MKN-45,经G418筛选后RT-PCR鉴定。结果:FHIT基因在人胃癌细胞系BGC-823中呈阳性表达,在MGC-803、MKN-45细胞系中呈阴性表达。FHIT基因cDNA正确克隆到真核细胞表达载体pcDNA3.1中,并成功转染FHIT基因mRNA阴性表达人胃癌细胞系MKN-45。结论:FHIT基因在不同类型人胃癌细胞系中表达各异。成功构建pcDNA3.1-FHIT,并转染到FHIT基因mRNA阴性表达人胃癌细胞系MKN-45,使其FHIT基因阳性表达。
Objective: To detect the expression of FHIT gene mRNA in human gastric cancer cell lines and to construct the eukaryotic expression vector pcDNA3.1-FHIT. Methods: The expression of FHIT gene mRNA in three different types of human gastric cancer cell lines was detected by RT-PCR. The eukaryotic expression plasmid pcDNA3.1-FHIT was constructed. The recombinant plasmid was identified by restriction enzyme digestion, PCR amplification and DNA sequence analysis , And then transfected into FHIT gene negative-expressing human gastric cancer cell line MKN-45 by lipofectamine. After screening by G418, the expression of MKN-45 was identified by RT-PCR. Results: FHIT gene was positively expressed in human gastric cancer cell line BGC-823 and negative in MGC-803 and MKN-45 cell lines. The FHIT gene cDNA was cloned into eukaryotic expression vector pcDNA3.1 and transfected into human gastric cancer cell line MKN-45 with FHIT mRNA. Conclusion: FHIT gene is expressed differently in different types of human gastric cancer cell lines. The recombinant plasmid pcDNA3.1-FHIT was successfully constructed and transfected into human gastric cancer cell line MKN-45 with FHIT mRNA.