摘要
目的 :克隆小鼠Notch1胞外段高抗原区编码基因 ,并进行原核表达及蛋白纯化 ,为制备mNotch1的单克隆抗体做准备。方法 :计算机分析小鼠Notch1全长 ,PCR扩增其胞外段高抗原区编码基因 ,克隆入载体T -vector进行序列测定 ,然后将测定正确的片段连入GST融合表达载体pGEX - 4T - 1,得到pG -NEF ,转化宿主菌DH5α ,经IPTG诱导 ,SDS -PAGE分析 ,以及新生条带表达形式分析以后 ,用GST亲和层析柱纯化。结果 :小鼠Notch1分子胞外段近膜处约 170个氨基酸区域抗原性较高 ,PCR扩增得到大小约 5 0 0bp的特异性片段 ,序列测定结果与文献报导的完全一致 ;原核表达产物大小约Mr 43× 10 3 ,以包涵体形式存在 ,约占总蛋白的2 5 % ,纯化后目的蛋白含量 >95 %。结论 :成功地进行了小鼠Notch1胞外段高抗原区编码基因的基因克隆、原核表达及蛋白纯化。
AIM: To clone gene encoding extra-cellular fragment with strong antigenicity of mNotch1 (mouse Notch1), express it in E coli . and purify the protein for preparation of anti-mNotch1 mcAb. METHODS: Full length of mNotch1 was analyzed with computer. The gene encoding extra-cellular fragment with strong antigenicity was amplified by PCR. The PCR product was inserted into T-vector for sequencing firstly, then inserted into pGEX-4T-1 for GST fusion expression vector pG-NEF. E.coli transformed with pG-NEF was induced with IPTG. The induced product was analyzed in SDS-PAGE and purified by GST-affinity-chromography method after protein location analysis. RESULTS: A fragment (about 170 amino acids) adjoining the trans-membrane region owned higher antigenicity in comparing to other extra-cellular parts of mNotch1. The PCR product was a unique band about 500bp. The sequencing result coincided the published sequence completely. The targeted protein expressing in E.coli as inclusion body was about M r 43×10 3. It accumulated up to 25% of the total protein of E.coli . After purification more than 95% of total protein were targeted protein. CONCLUSION: The gene cloning, protein expression and purification of extra-cellular fragment with strong antigenicity of mNotch1 were performed successfully.
出处
《牙体牙髓牙周病学杂志》
CAS
2002年第3期145-148,共4页
Chinese Journal of Conservative Dentistry
关键词
NOTCH1
基因克隆
原核表达
蛋白纯化
notch1
clone gene
targeted protein expressing
protein purification
