人核糖体蛋白S13(RPS13)编码基因的克隆及其正反义核酸转染胃癌细胞的初步研究

目的：扩增人核糖体蛋白S13（RPS13）编码基因序列，构建其正，反义真核表达载体，分别转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，以进一步研究RPS13基因与胃癌多药耐药的关系。方法：采用RT-PCR法扩增RPS13cDNA片段编码区全长序列，利用DNA重组技术的建正，反义真核表达载体，经脂质体介导转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，筛选正，反义基因稳定转染细胞，RNA斑点杂交法检测正，反义稳定转染细胞mRNA水平的变化。结果：从高表达RPS13的SGC7901/VCR耐药细胞中提取细胞总RNA，RT-PCR法成功扩增了RPS13cDNA片段编码区全长序列，并经测序证实；目的基因因按正，反两个方向亚克隆至真核表达载体pcDNA3.1( ),并经酶切鉴定证实；经脂质体介导将正义真核表达载体转染SGC7901细胞，反义真核表达载体转染SGC7901/VCR细胞，G418筛选获得稳定转染细胞；RNA斑点杂交试验证实；正义稳定转染细胞RPS13mRNA水平上调，反义稳定转染细胞RPS13mRNA水平下调。结论：成功克隆了人RPS13编码基因序列，并构建了其正，反义真核表达载体，在胃癌细胞系SGC7901及长春新碱耐药细胞SGC7901/VCR中得到稳定转染细胞，正义转染细胞中RPS13mRNA表达明显增加，反义转染细胞中表达明显受抑。

Cloning RPS13 Encoding gene and Transfecting Sense Antisense Nucleic Acid into Gastric CDancer Cell

Objective:To obtain human ribosomal protein S13(RPS13)encoding gene,con-struct its sense?antisense eukaryotic vectors and transfect into gastric cancer cell line SGC7901and vincristine-resistant gastric cancer cell variant SGC7901/VCR in order to describe the associ-ation of RPS13with MDR phenotypes of SGC7901/VCR cells.Methods:RPS13cDNA was am-plified by RT-PCR.The sense and antisense eukaryotic expression vectors were constructed using DNA recombination technique.The recombinant sense and antisense vectors were then introduced into SGC7901and SGC7901/VCR cells respectively by liposome.Stable clones were obtained by screening with G418.RNA dot blotting assay and it was used to verify the changes of mRNA ex-pression in stable clones.Results:Full-length cDNA encoding RPS13was amplified by RT-PCR from Vincristine-resistant SGC7901/VCR cells.The PCR product was sequenced and verified for the proper encoding.The sense,antisense eukaryotic expression vectors were constructed by direct cloning the target gene into eukary otic expression vector pcDNA3.1(+),and subsequently confirmed by en donuclease digestion.Using liposome mediated method,gastric cancer cell line SGC7901was transfected with sense re combinant vector,while Vin cristine-resistant SGC7901/VCR cells were transfected with antisense re combinant vector.Stable clones ob tained after G418screening.RNA dot blotting assay suggested that mRNA level of the RPS13was up-regu lated in the sense recom-binant vector transfected cell,and down-regulated in the antisense recombinant vec tor transfected cells.Conclusion:hRPS13encoding gene is successfully amplified with RT-PCR and its sense,an tisense eukaryotic vectors are successfully constructed.The RPS13sense RNA can be ex-pressed steadily in human gastric cancer cell line SGC7901and higher RPS13mRNA level,while RPS13antisense RNA can be expressed steadily in Vincristine-resistant gastric cancer cell variant SGC7901/VCR and lower RPS13mRNA level.