[Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity]
Zhen-Ni Ma1, Jing Ling2, Jie Yin3
1Jiangsu Provincial Institute of Hematology，Key Laboratory of Thrombosis and Hemostasis of Ministry of Health，The First Affiliated Hospital of Soochow University，Collaborative Innovation Center of Hematology，Soochow University,Suzhou 215000, Jiangsu Province，China,E-mail: windfr@163.com.
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.
