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目的构建含尿激酶纤溶酶原激活剂(urokinase plasminogen activator,uPA)裂解位点的人可溶性凋亡诱导配体(soluble related apoptosis inducing ligand,sTrail)基因的原核载体,表达并纯化其融合蛋白sTrail-uPA。方法 RT-PCR法克隆sTrail基因后,经引物延伸法构建his-uPA-sTrail融合基因并亚克隆至原核表达载体pET-32a中,诱导其表达蛋白并将其纯化,肠肽酶(enterokinase,EK)切与再次纯化回收该蛋白,Western blot检测其抗原性。结果表达载体经诱导成功获约38×103含载体表达标签(Trx)的融合蛋白,EK酶切该蛋白获约19.5×103的目的蛋白uPA-sTrail。检测表明,该目的蛋白具人Trail抗原性。结论成功克隆uPA-sTrail融合基因并构建至原核表达载体,并获约19.5×103的融合蛋白,且该蛋白具人Trail抗原性。
OBJECTIVE: To construct a prokaryotic vector containing sTrail gene of urokinase plasminogen activator (uPA) cleavage site and to express and purify the fusion protein sTrail -uPA. Methods The sTrail gene was cloned by RT-PCR and his-uPA-sTrail fusion gene was constructed by primer extension and subcloned into the prokaryotic expression vector pET-32a. The recombinant protein was induced and purified. Enterokinase (EK ) Cut and purified again to recover the protein, Western blot detection of antigenicity. Results The expression vector was successfully induced with about 38 × 103 fusion protein containing vector expression tag (Trx). EK enzyme digestion of the protein obtained about 19.5 × 103 of the target protein uPA-sTrail. The test showed that the target protein has human Trail antigenicity. Conclusion The uPA-sTrail fusion gene was successfully cloned and constructed into prokaryotic expression vector. The fusion protein was about 19.5 × 103, and the protein was human Trail antigen.