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目的探讨差异表达基因组织蛋白酶B在大鼠矽肺模型中的作用。方法对以往经抑制消减杂交筛选得到的一条编号为ES110708的差异表达序列标签(EST)作为种子序列,通过电子克隆进行延伸,并在大鼠矽肺模型中用RT-PCR方法进行验证。结果筛选得到的大鼠矽肺差异表达EST经电子克隆延伸得到了含有完整开放读码框架(ORF)的cDNA序列,长度为1977bp,比原来的EST片段(172bp)延伸了1805 bp。其ORF预测为1020 bp,符合Kozak规则。将ORF序列进行同源性检索,其与序列号为NM022597.2的cDNA部分序列完全同源,说明所测ORF属于序列NM022597.2。该序列代表的基因是组织蛋白酶B。经RT-PCR验证,实验组大鼠肺组织Cathepsin B mRNA表达量明显上调。15 d、30 d、60 d时,其吸光度(A)值分别为对照组的4.133、6.639、4.981倍,差异具有统计学意义(P<0.05)。结论电子克隆能有效延伸EST序列直至全长cDNA序列,差异表达基因组织蛋白酶B可能参与了矽肺的发生与发展。
Objective To investigate the role of differentially expressed cathepsin B in rat silicosis model. Methods A differentially expressed sequence tag (EST) numbered ES110708, which was screened by suppression subtractive hybridization, was used as a seed sequence and extended by electronic cloning and verified by RT-PCR in silicotic rat model. Results The differentially expressed ESTs of silicotic lung in rats were cloned and extended by electronic cloning. The cDNA sequence of complete open reading frame (ORF) was obtained and extended for a length of 1977 bp, which was 1805 bp longer than the original EST fragment (172 bp). The ORF predicted 1020 bp, in line with Kozak rules. Homology search of ORF sequence was performed, which was completely homologous with the cDNA partial sequence of sequence number NM022597.2, indicating that the measured ORF belongs to sequence NM022597.2. The gene represented by this sequence is cathepsin B. The results of RT-PCR showed that the expression of Cathepsin B mRNA in the lung tissue of experimental group was significantly up-regulated. The absorbance (A) value of control group was 4.133,6.639 and 4.981 respectively at 15 d, 30 d and 60 d, the difference was statistically significant (P <0.05). Conclusion Electronic cloning can effectively extend the EST sequence until the full-length cDNA sequence, and differential expression of cathepsin B may be involved in the occurrence and development of silicosis.