大鼠矽肺相关基因组织蛋白酶B的电子克隆与验证  

In silico cloning and verification of cathepsin B related to silicosis in rats

作　　者：王朝辉[1] 马小兵[1] 侯文娜[2] 徐慧蓉[3] 朱兰[1] 伊雪[1] 

机构地区：[1]河北联合大学基础医学院病理学系,河北唐山063000 [2]唐山市眼科医院病理科,河北唐山063000 [3]淄博市中心医院病理科,山东淄博255000

出　　处：《中国工业医学杂志》2014年第2期93-96,F0003,共5页Chinese Journal of Industrial Medicine

基　　金：河北省科学技术研究与发展计划项目(09276192D)

摘　　要：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 study the role of Cathepsin B, a differentially expressed gene in pathogenesis of silicosis using rats model. Methods The preliminary study using suppression subtraetive hybridization technique already screened out hundreds of silicotic rats related genes, including many expessed sequence tags （ESTs） , then a full long eDNA was got by in silieo cloning from a differentially expressed EST （ No ES110708 ）, and verified by RT-PCR in lung tissue of rats with silicosis. Results A full long eDNA as 1977 bp （base pairs） containing complete open reading frame （ORF） was got by in silico cloning from the seed sequence ES110708, and 1805 base pairs longer than its original EST （long as 172 base pairs）. Its ORF was predicted as 1020 bp that conformed to Kozak rules. By ORF sequenced homology search online, its sequence was homologous completely with part of eDNA sequence （ serial number for NM022597.2）. The eDNA sequence belongs to eathepsin B, and its mRNA showed obvious over-expression in lung tissues of silicosis rats. The expressions of cathepsin B mRNA in lung tissues of silicosis rats at 15 d, 30 d, and 60 d were 4. 133, 6. 639, and 4. 981 times higher than that in control group respectively （P 〈 0. 05 ）. Conclusion A full long eDNA can be got from a EST by in silico cloning ; the differentially expressed gene cathepsin B maybe involves in the occurrence and development of silicosis.

关 键 词：组织蛋白酶B 矽肺 差异表达基因 电子克隆 

分 类 号：R99[医药卫生—毒理学]