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目的构建表达人干扰素-α2b(hIFN-α2b)蛋白的双歧杆菌工程菌,并验证靶蛋白的表达水平。方法人工合成hIFN-α2b基因,插入双酶切的pBAD质粒,构建重组质粒pBAD-IFN。制备双歧杆菌感受态,用pBAD-IFN电转化后PCR鉴定、筛选阳性克隆,筛选的阳性双歧杆菌克隆在L-阿拉伯糖诱导下表达靶蛋白,并进行Western blot检测。结果测序和双酶切鉴定表明成功构建重组质粒pBAD-IFN,Western blot检测证明氨苄青霉素筛选出的该质粒转化双歧杆菌阳性克隆经L-阿拉伯糖诱导表达分子质量单位为18ku的hIFN-α2b蛋白。结论 hIFN-α2b表达载体pBAD-IFN转化双歧杆菌后可表达hIFN-α2b蛋白。
Objective To construct an engineered Bifidobacterium expressing human interferon-α2b (hIFN-α2b) protein and verify its target protein expression. Methods Human hIFN-α2b gene was synthesized and inserted into double-digested pBAD plasmid to construct recombinant plasmid pBAD-IFN. The competent Bifidobacterium was prepared and transformed into pBAD-IFN. The positive clones were screened by electroporation with pBAD-IFN. The positive Bifidobacterium positive clones were selected and expressed by L-arabinose. The target proteins were detected by Western blot. Results Sequencing and restriction enzyme digestion showed that recombinant plasmid pBAD-IFN was successfully constructed. Western blot analysis confirmed that ampicillin-positive plasmids were transformed into Bifidobacterium positive clones, and induced by L-arabinose to express hIFN-α2b protein with a mass unit of 18 ku . Conclusion hIFN-α2b protein can be expressed after transforming hIFN-α2b expression vector with Bifidobacterium.