摘要
目的:克隆NK4基因,构建其重组真核表达载体,并观察其在Raji细胞中的表达。方法:从人肝组织中提取总RNA,行RT-PCR获得NK4基因cDNA,与载体pVITRO2-mcs构建重组真核表达载体,转染Raji细胞。潮霉素B筛选后,采用实时荧光定量PCR、ELISA、细胞免疫组化和半固体培养等方法检测NK4基因在Raji细胞的表达,筛选稳定表达的细胞株。结果:NK4基因获成功克隆并构建了重组真核表达载体pVITRO2-mcs-NK4;转染NK4基因后的Raji细胞经RT-PCR发现存在特异性DNA片段。NK4基因在Raji细胞可稳定表达NK4mR-NA和蛋白,且可抑制Raji细胞的增殖、迁徙和侵袭。结论:NK4基因成功克隆并构建了重组真核表达载体,转染Raji细胞后表达稳定。
AIM: To clone NK4 gene and to construct recombinant eukaryotic expression vector for observing its expression in transfected Raji cells. METHODS: Total RNA was extracted from human hepatic tissue. NK4 gene cDNA was amplified by RT - PCR, and then cloned into vector pVITRO2 - mcs to construct the recombinant eukaryotic expression vector pVITRO2 - mcs - NK4. Raji cells were transfected by recombinant vector pVITRO2 - mcs - NK4 and screened by homomycin B. The stable strain of NK4 gene expression was screened by real - time fluorescent quantitative PCR, ELISA, immunocytohistochemistry and semisolid culture. RESULTS: The specific DNA fragment was detected by RT- PCR in Raji cells transfected with NK4 gene. The transfected Raji cells expressed NK4 mRNA and protein stably, which inhibited Raji cell proliferation, metastasis and invasion. CONCLUSION: NK4 gene is cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2 - mcs - NK4 successfully. NK4 gene in Raji cells expresses stably.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2009年第5期970-975,共6页
Chinese Journal of Pathophysiology
基金
浙江省医药卫生科学基金资助项目(No2003B172
No.2007A175)
宁波市医药卫生科学基金资助项目(No.2003079)
宁波市科技计划资助项目(No.2007C10065)