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目的 检测人小肠黏膜防御素 5基因 (Alphadefensin 5 ,DEFA5)的表达 ,为从分子水平上探讨小肠黏膜抗菌防御机制提供实验依据。方法 从 2例新鲜尸体和 2例右半结肠癌患者的回肠部位取小肠黏膜提取总RNA ,通过RT PCR扩增出DEFA5的两个外显子序列 ,比较其个体间的差异 ;沿小肠轴间隔 30cm取新鲜尸体小肠黏膜提取总RNA ,仍通过RT PCR扩增编码HD 5的阅读框序列 ,比较HD 5沿肠轴的表达趋势。结果 DEFA5在个体间的表达是一致的 ,通过RT PCR获得的外显子序列大小为 4 5 1bp ,且碱基序列与GenBank中的DEFA5标准序列相一致 ;通过PCR获得的编码HD 5的阅读框序列为 30 3bp ,其中 ,酶切位点 12bp ,保护性碱基 6bp ,终止子 3bp ,编码HD 5的序列 2 82bp(94AA) ;沿着空肠到回肠的肠轴方向 ,DEFA5在空回肠黏膜均有表达 ,至回肠末端最强 ,并有逐渐增强的趋势。结论 DEFA5基因的表达在个体间是保守的 ,而且沿着空肠到回肠的肠轴方向在mRNA水平的表达有逐渐增加的趋势 ,提示HD 5作为天然免疫物质在小肠黏膜屏障功能的维护中起着十分重要的作用。此外 ,本实验获得的HD 5阅读框序列为进一步重组表达HD 5奠定了基础。
Objective To detect the expression of human defensin 5 gene (DEFA5), and to provide experimental evidence for exploring the antimicrobial defense mechanism of small intestinal mucosa at the molecular level. Methods Total RNA was extracted from small intestine mucosa of 2 fresh corpses and 2 right colon cancer patients. Two exon sequences of DEFA5 were amplified by RT-PCR, and their inter-individual differences were compared. Total RNA was extracted from fresh cadaver intestinal mucosa by 30cm, and the sequence encoding HD5 was still amplified by RT-PCR. The expression of HD5 along the intestinal axis was also compared. Results The expression of DEFA5 was consistent among individuals. The size of exon sequence obtained by RT PCR was 45 lbp, and the nucleotide sequence was consistent with the DEFA5 standard sequence in GenBank. The reading frame of HD5 encoding by PCR The sequence was 30bp, in which the enzyme digestion site 12bp, the protective base 6bp, the terminator 3bp, the sequence encoding HD5 2 82bp (94AA); along the jejunum to ileum in the intestinal axis direction, DEFA5 in the ileum mucosa Expression, to the strongest ileum, and a gradual increase in the trend. CONCLUSIONS: The expression of DEFA5 gene is conserved among individuals and tends to increase at the mRNA level along the jejunum to the ileum. This suggests that HD 5 acts as a natural immune substance in the maintenance of intestinal mucosal barrier function Very important role. In addition, the HD5 reading frame obtained in this experiment laid the foundation for the further recombinant expression of HD5.