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目的:获得大量重组大鼠β细胞素并检测其活性。方法:用PCR法从大鼠肾组织中扩增534bp的β细胞素基因片段,并按读框克隆到原核表达载体pET28a(+)中。以构建的重组质粒pET28a-rBTC转化大肠杆菌BL-21(DE3)菌株,在IPTG诱导下表达β细胞素蛋白,表达产物用SDS-PAGE和Westernblot检测。采用镍柱亲和层析法纯化目的蛋白。结果:经IPTG诱导后,在大肠杆菌中可表达相对分子质量Mr为20000的目的蛋白。目的蛋白主要以包涵体的形式表达;表达量约占菌体蛋白总量的20%～30%。表达的目的蛋白与抗His标签抗体具有良好的反应性。结论:大鼠β细胞素基因在PET表达系统中得到高效表达;蛋白复性后能有效地促进NIH3T3细胞的体外增殖。
Objective: To obtain a large number of recombinant rat β cytokines and test their activity. Methods: 534bp β-cytidylic gene fragment was amplified by PCR from rat kidney and cloned into prokaryotic expression vector pET28a (+) in frame. The recombinant plasmid pET28a-rBTC was transformed into Escherichia coli BL-21 (DE3) strain to express the β-cytidycine protein under the induction of IPTG. The expressed product was detected by SDS-PAGE and Western blot. The target protein was purified by nickel column affinity chromatography. Results: After induced by IPTG, the target protein with molecular weight of Mr 20000 could be expressed in Escherichia coli. The target protein is mainly expressed in the form of inclusion body; the expression level accounts for about 20% -30% of the total bacterial protein. The expressed protein has good reactivity with anti-His-tag antibody. CONCLUSION: The rat β-cytokine gene is highly expressed in the PET expression system and the protein renaturation can effectively promote the proliferation of NIH3T3 cells in vitro.