【摘要】 以高效原核表达载体pCold TF为载体骨架,应用PCR、限制性酶切、连接等分子生物学方法,将pCold TF中的六聚组氨酸(His-Tag)序列替换为限制性酶切位点SpeⅠ序列ACTAGT,构建不含His-Tag序列的新载体pL118.以此载体表达蛋白时,通过PCR引物在目的基因3′端添加His-Tag以利于融合蛋白的纯化以及融合蛋白中的Trigger Fac-tor(TF)助溶蛋白的去除.应用pL118对人膜蛋白CD81进行原核表达与纯化,并使用Fac-tor Xa蛋白酶去除CD81融合蛋白中的TF助溶蛋白,获得3′端含有His-Tag的CD81蛋白.人膜蛋白CD81的表达纯化,为CD81靶向药物筛选、抗体制备及深入研究CD81的功能奠定了基础.pL118载体的构建以及CD81蛋白的表达纯化为表达困难的基因实现原核表达提供了一种新的思路和方法.更多还原

【Abstract】 Based on prokaryotic expression vector pCold TF,a new vector pL118 was constructed by replacing the His-Tag sequence of the original vector with the restriction enzyme site of Spe I in molecular biological methods.His-Tag sequence was introduced to the 3′ end of target gene through PCR primer,and this method facilitated the expression of fusion protein and the removal of solubilization tag from the target protein.The fusion protein human CD81-Trigger Factor(TF) was expressed and purified.Human CD81 protein was obtained by removing the TF solubilization tag by proteinase Factor Xa.The purified human CD81 protein laid the foundation for the selection of targeted drugs,making antibody and exploring the function of CD81.The construction of the new expression vector pL118 and the expression and purification of human CD81 protein have provided a new method for obtaining insoluble protein based on the prokaryotic expression system.更多还原