摘要
目的克隆小鼠CXCR3基因,将其胞外N末端在大肠杆菌中进行可溶性表达并用于抗体制备。方法采用RT-PCR技术,从Balb/c小鼠脾细胞中扩增出编码CXCR3的全长序列,将胞外N末端亚克隆入DsbA融合表达载体,转化大肠杆菌BL21(DE3),诱导表达后进行分离纯化,然后免疫家兔制备多克隆抗体,并用趋化小室法检测抗体对IP-10趋化小鼠T细胞作用的影响。结果获得编码小鼠CXCR3的cDNA序列,其胞外N末端与DsbA融合后能在大肠杆菌中可溶性表达,表达量达40%,其抗体可显著抑制IP-10对小鼠T细胞的趋化作用。结论获得小鼠趋化因子受体CXCR3胞外N末端融合蛋白及其抗体,抗体具有在体外抑制T细胞趋化的作用。
Objective To clone the gene of mouse chemokine receptor CXCR3, and to express extracellular N-terminal domain of CXCR3 in E.coli for antibody production. Methods mCXCR3 gene was cloned from splenocytes of Balb/c mouse by RT-PCR. Extracellular N-terminal domain of mCXCR3 was subcloned into DsbA fusion protein expression vector, and then expressed in E.coli . The purified target protein was used to generate rabbit antiserum. The effect of the antiserum on IP-10-induced lymphocyte chemotaxis was measured by Boyden chamber assay. Results The fusion protein of CXCR3 extracellular N-terminal domain and DsbA was expressed in E.coli BL21 (DE3). Soluble target protein amounted to 40% in total bacteria protein. The purified antiserum showed inhibiting effect on IP-10-induced lymphocyte chemotaxis. Conclusion Recombinant protein of the extracellular N-terminal domain of mCXCR3 is obtained. The specific antiserum can remarkably inhibit the effect of IP-10 on lymphocyte chemotaxis in mouse.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第3期197-200,共4页
Immunological Journal
