人白血病细胞系J6-1变异M-CSF的表达及受体结合活性研究

Expression of Mutant M-CSF from Human Leukemic Cell Line J6-1 and the Binding Activity for Its Receptor

目的 :从人白血病细胞系J6 1克隆并表达变异的M CSF的功能区 ,研究其受体结合活性。方法 :RT PCR克隆muM CSF ,并在大肠杆菌BL2 1trxB(DE3)、pET32c(+)系统中表达 ;镍柱鏊合层析 ,抗体亲和层析纯化。ELISA法测定其受体结合活性和解离常数 ,并测定对J6 1细胞增殖刺激的活性。结果 :muM CSF可在本文实验系统呈可溶性表达 ,纯化产物具有M CSF受体的结合活性 ,解离常数为 3.7nmol/L低于正常M CSF ,且刺激细胞体外增殖能力大于正常M CSF。结论 :从人白血病细胞系J6 1克隆的muM CSF能在BL2 1,pET32c(+)系统表达 。

Objective: To clone and express functional part of mutant M CSF (muM CSF) from human leukemic cell line J6 1 and investigate its Kd for dissociation and biological activity on the proliferation of J6 1. Method: Functional part of muM CSF was cloned by RT PCR and inserted into pET32c(+) and expressed in E.coli BL21 trx B (DE3). The recombinant protein was purified through Ni 2+ affinity column and antibody linked affinity column. ELISA was performed to define the Kd of the muM CSF to its receptor. Colony formation assay was performed to test its effects on the proliferation of J6 1. Results: The protein was purified and its Kd to the receptor was 3.7 nmol/L. muM CSF showed elevated proliferation stimulating potential than normal M CSF. Conclusion: muM CSF could be expressed in the pET32c(+), BL21 system and the muM CSF showed elevated proliferation promoting ability as to normal M CSF.