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目的环氧合酶-2与肿瘤发生的关系已成为肿瘤研究的新热点之一,本研究旨在获得 hCOX-2编码基因序列,构建其正、反义真核表达载体,并用反义重组载体转染 COX-2高表达的胃癌细胞,以进一步研究其生物学作用及其在肿瘤发生中的作用机制。方法按文献报道的hCOX-2核苷酸序列设计合成 PCR 引物,利用总 RNA 抽取试剂盒从 COX-2高表达的培养的人胃癌细胞系 SHC7901中提取细胞总 RNA,反转录合成 cDNA,再用PCR 方法扩增目的基因序列,PCR 产物经 DNA 序列测定证实后,利用引物5端引入的 EcoRⅠ,Xba Ⅰ酶切位点,通过粘端连接,将所获目的基因分别按正、反两个方向定向克隆入真核表达载体 pcDNA3.1中,对两种重组质粒分别进行相应的酶切鉴定,采用脂质体介导的方法用 COX-2反义核酸转染 SGC7901细胞,经 G418筛选后,随机挑选细胞克隆,通过 RT-PCR 检测COX-2 mRNA 水平的变化。结果应用 RT-PCR 反应扩增获得大小约1.9kb 的特异性片段,经 DNA 序列分析,证实与文献报道的 hCOX-2编码区序列一致,重组正义表达载体 pcDNA3.1/hCOX2(+)用 EcoRⅠ,Xba Ⅰ双酶切后,产生大小约5.4kb,1.9kb 的片段;重组反义表达载体 pcDNA3.1/hCOX2(-)通过 PstⅠ酶切,产生大小约4.5kb,1.5kb 与1.3kb 的片段,与预期结果相符,转染细胞经筛选后,随机挑选、扩增了3个细胞克隆,其中1个克隆稳定表达 COX-2反义核酸,RT-PCR 提示其 COX-2 mRNA 水平显著下降.结论成功克隆了 hCOX-2编码基因序列,并构建了其正、反义真核表达载体.反转录 PCR 是克隆基因的简便、有效方法.为扩增高 GC 含量的 cDNA 模板,可在 PCR 反应体系中加入适量的 DMSO,并采用较高退火温度.在 PCR 引物中加入限制性内切酶位点,可以方便地实现基因的定向克隆.COX-2反义核酸在胃癌细胞系 SGC7901中得到稳定转染,转染细胞 COX-2mRNA 表达显著受抑制.
The relationship between cyclooxygenase-2 and tumorigenesis has become a new hot spot in tumor research. This study aims to obtain the hCOX-2 coding gene sequence, construct its positive and antisense eukaryotic expression vectors, and use antisense recombinant vectors. The COX-2 highly expressed gastric cancer cells were transfected to further study its biological role and its mechanism in tumorigenesis. Methods PCR primers were designed and synthesized according to the nucleotide sequence of hCOX-2 reported in the literature. Total RNA was extracted from cultured human gastric cancer cell line SHC7901 highly expressed by COX-2 using total RNA extraction kit, and cDNA was reverse transcribed and synthesized. The PCR product was used to amplify the target gene sequence. PCR products were confirmed by DNA sequence analysis. The EcoRI and Xba I restriction sites introduced at the 5′ end of the primers were used to bind the obtained target gene to the positive and negative domains. Directionally directionally cloned into the eukaryotic expression vector pcDNA3.1. The two recombinant plasmids were identified by restriction endonuclease digestion. The SGC7901 cells were transfected with COX-2 antisense nucleic acid using a liposome-mediated method and screened by G418. The cell clones were randomly selected and the level of COX-2 mRNA was detected by RT-PCR. RESULTS: RT-PCR was used to amplify the specific fragment of about 1.9 kb. DNA sequence analysis confirmed that the hCOX-2 coding sequence was consistent with that reported in the literature. Recombinant sense expression vector pcDNA3.1/hCOX2(+) was used as EcoRI. After Xba I double digestion, a fragment of approximately 5.4 kb and 1.9 kb in size was generated; the recombinant antisense expression vector pcDNA3.1/hCOX2(-) was digested with PstI to yield fragments of approximately 4.5 kb, 1.5 kb and 1.3 kb in size. , Consistent with the expected results, after transfected cells were selected, three cell clones were randomly selected and amplified, and one of the clones stably expressed COX-2 antisense nucleic acid, and the level of COX-2 mRNA was significantly decreased by RT-PCR. Conclusion The hCOX-2 coding gene sequence was successfully cloned and its positive and antisense eukaryotic expression vectors were constructed. Reverse transcription PCR is a simple and effective method for gene cloning. It is a high GC content cDNA template for PCR amplification. An appropriate amount of DMSO was added to the reaction system and a higher annealing temperature was used. Adding restriction enzyme sites into the PCR primers facilitated the directional cloning of genes. COX-2 antisense nucleic acids were obtained in the gastric cancer cell line SGC7901. Stable transfection, significant expression of COX-2 mRNA in transfected cells Suppression.