鸡破骨细胞分化因子(chRANKL)的克隆、表达和活性分析  

Cloning, Expression and Bioactivity Analysis of Chicken Receptor Activator of NF-κB Ligand(chRANKL )

作　　者：王艳[1] 姚静[1] 张风荣[1] 侯加法[1] 

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《农业生物技术学报》2008年第1期32-36,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30671546);江苏省自然科学基金(No.BK2004099);高校博士点基金项目(No.20040307036)

摘　　要：以鸡(Gallus domesticus)成骨细胞总RNA为模板,利用RT-PCR和SOE-PCR技术体外扩增鸡破骨细胞分化因子(RANKL)基因,将PCR产物克隆至组氨酸标签的融合蛋白表达载体pET-32a(+),在异丙基-β-D硫代半乳糖苷(IPTG)诱导下,实现了chRANKL的有效表达,表达产物纯化后稀释成不同浓度梯度作用成熟破骨细胞观察其生物学活性。结果表明,凝胶电泳显示PCR扩增产物的长度为1200bp左右,插入片段与GenBank上报道的鸡RANKL序列完全一致。重组表达载体转化BL21后经IPTG诱导获得大小约为64kD的重组蛋白,Western印迹表明重组蛋白能与抗组氨酸抗体相结合,具有特异反应性;并且纯化后的蛋白能刺激成熟破骨细胞,使骨吸收指数呈剂量依赖性增加,表明具有一定的活性。Using reverse transcription polymerase chain reaction （RT-PCR） and gene splicing by overlap extension （SOE-PCR）, the DNA sequence encoding the chicken＇s receptor activator ofNF -κB ligand （RANKL） was amplified, and then cloned into the expressing plasmid pET32a（＋） with His-tag, and was highly expressed in Echerichia coli. Then the purified protein was added in primary cultures of chicken osteopclasts to observe its bioactivity. The results showed that the size of PCR product was 1 200 bp which was consistent with the expected one, and the relative molecular weight of induced protein was 64 kD. Western blot indicated that the induced protein could react with anti-His antibody. It was also found that the induced protein could stimulate mature chicken osteoclasts to resorb bone.

关 键 词：鸡破骨细胞分化因子(chRANKL) 基因克隆 原核表达 

分 类 号：S188[农业科学—农业基础科学]