Spacer区蛋白对ADAMTS13活性影响的研究

Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity

目的:本研究旨在获得血管性血友病因子裂解酶ADAMTS13中重组的spacer区蛋白,进一步探讨该区域对ADAMTS13功能发挥的作用。方法:以ADAMTS13全长质粒为模板,构建含spacer区基因序列的原核表达质粒,转染大肠杆菌,低温(30℃)IPTG诱导表达,收集超声上清并鉴定。并大规模诱导表达,利用Ni-NTA琼脂糖柱,梯度咪唑淋洗法纯化蛋白,应用SDS-PAGE和Western blot法鉴定纯化产品纯度和免疫学活性。利用重组蛋白与免疫性调节的血栓性血小板减少性紫癜(iTTP)患者血浆孵育,检测血浆中ADAMTS13的活性变化。结果:成功构建能表达spacer区的原核表达质粒并能有效表达蛋白。Western blotting结果显示,抗6×His抗体和抗人ADAMTS13抗体(针对Gln34-Trp688)分别能与重组蛋白在15 kDa处显单一条带。功能实验表明,该重组蛋白能够有效逆转iTTP患者中被自身抗体抑制的ADAMTS13酶活性。结论:重组蛋白具有较好的免疫原活性和纯度,为进一步研究spacer区对iTTP的发病机理提供了实验依据。

Objective:To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease(ADAMTS13),and further study its biological function in ADAMTS13.Methods:The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13,and then transfected into E coli.,following the induction of IPTG with the low temperature(30℃).The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole.The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively.By Adding the recombinant protein to the plasma of immune-mediated thrombotic thrombocytopenic purpura(iTTP)patients,the activity of ADAMTS13 was tested.Results:The prokaryotic expression vector was successfully constructed and the protein of spacer domain with the high purity was obtained.Western blot showed that the recombinant fragment could both react with monoclonal antibody against 6×His and polyclonal sheep IgG against ADAMTS13(Gln34-Trp688).The protein formed a main lane at the position of 15 kDa with SDS-PAGE.It was demonstrated that the recombinant protein could efficiently elevate the ADAMTS13 activity in plasma of iTTP patients to reach normal level by functional experiment.Conclusion:The recombinant protein has high purity and immune activity,which provides the experimental basis for further research on mechanism of iTTP involved in spacer domain.