梅花鹿colX基因的克隆及原核表达

Cloning and prokaryotic expression of sika deer colX gene

提取健康梅花鹿鹿茸肥大软骨细胞总RNA,以RT-PCR获得colX基因2 038 bp的完整编码序列,将其克隆入载体pMD18-T中,经限制性内切酶和质粒PCR分析,并测序证实的阳性重组子与表达载体pET-28a(+)连接,转化大肠杆菌BL21(DE3)。以IPTG诱导,经SDS-PAGE及Western-blot分析表明,获得相对分子质量为65 000的colX蛋白表达带,与预期相符,表明成功获得梅花鹿colX基因蛋白。

The total RNA was extracted from normal sika deer antler hypertrophic chondrocyte and used as a template for RT-PCR. A 2 038 bp fragment containing the whole length gene coding region of protein colX was obtained and cloned into pMD18-T vector. The recombinant which had analysied by restriction endonucleases and PCR was sequenced to confirm its identity. The right recombinant was ligated with vector pET-28a（＋） and transformed into E. coli BL21 （DE3）. After being induced by IPTG, SDS-PAGE and Western-blot analysis confirmed that the transformed E. coli BL21 could express colX proteins with the molecular weight of 65 000. The results showed that the sika deer colX protein was obtained successfully.