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G15γ5′是本组以往用mRNA差异显示法从γ干扰素预处理的小鼠腺癌细胞系MA891中分离出的cDNA片段,推测其代表的基因可能与肿瘤转移有关。为进一步研究其功能,采用cDNA未端快速扩增技术(RACE),以获取该片段5′端未知序列。用IFN-γ(200 U/ml)处理MA891细胞48 h,提取总RNA并用DNaseI纯化。根据G1575′端序列,设计合成3个基因特异性引物GSP1,GSP2和GSP3。以GSP1为引物合成cDNA第1链,用末端转移酶在cDNA第1链的3′端进行同聚物加尾,对加尾后的cDNA先后用GSP2和GSP3与锚定引物进行初级及巢式PCR扩增,最终得到一552 bp的扩增产物并将其克隆至pCR~(TM) I载体,经Northern印迹分析及序列分析证实,所得片段确实是G15T5′的5′端延续。
G15γ5′ is a cDNA fragment previously isolated from gamma interferon-pretreated mouse adenocarcinoma cell line MA891 by mRNA differential display in this group, and it is speculated that the gene represented may be involved in tumor metastasis. To further investigate its function, a rapid cDNA amplification technique (RACE) was used to obtain the unknown sequence at the 5′ end of the fragment. MA891 cells were treated with IFN-γ (200 U/ml) for 48 h, total RNA was extracted and purified with DNase I. According to the sequence of G1575′, three gene-specific primers GSP1, GSP2 and GSP3 were designed and synthesized. The first strand of cDNA was synthesized using GSP1 as a primer, homopolymer tailing was performed at the 3′ end of the first strand of cDNA using terminal transferase, and the tailed cDNA was primary and nested with GSP2 and GSP3 and anchor primers. After PCR amplification, a 552 bp amplified product was finally obtained and cloned into the pCRTM carrier. Northern blot analysis and sequence analysis confirmed that the resulting fragment was indeed the 5’ end of G15T5’.