人IFN-γ与GM-CSF融合基因重组BCG表达与鉴定

Expression and identification of recombinant Bacillus Calmette-Guerin fusion plasmid with construction of human IFN-γ and GM-CSF

目的构建人IFN-γ与GM-CSF融合基因的卡介苗(BCG)重组质粒,并在卡介苗中分泌表达人IFN-γ与GM-CSF蛋白。方法采用反转录PCR,分别获得人IFN-γ与GM-CSF基因编码序列的c DNA。将IFN-γ与GM-CSF基因进行PCR扩增,通过接头(Gly_4Ser)_3将其连接,形成融合基因,将融合基因插入到p MV261质粒HSP60启动子下游,构建一个分泌型的卡介苗-大肠杆菌穿梭表达质粒p MVGMI-γ。转化感受态细胞E.coli DH5α,在LB培养基平板上进行卡那霉素抗性选择,选择阳性克隆提取质粒后转化感受态BCG,用(NH_4)_2SO_4沉淀、分子筛层析柱Bio-Gel P-6 DG纯化目的蛋白,10%SDS-PAGE凝胶电泳检测纯化结果,Western-blot检测人IFN-γ与GM-CSF基因在r BCG中的表达。结果目的基因GM-CSF和IFN-γRT-PCR产物大小分别为461 bp和432 bp,与预期值一致。构建的重组质粒经双酶切、扩增及测序鉴定,融合基因GMI-γ(462+435-45=852 bp)正确插入载体,成功转化入BCG感受态细胞,且正确表达。结论融合基因GMI-γ修饰的r BCG构建成功并表达,为进一步探讨r BCG免疫活性及发展新型抗结核杆菌疫苗奠定了基础。

Objective To construct a recombinant Bacillus Calmette- Guerin（BCG） plasmid that combining theexpression of human IFN-γ and GM-CSF and determine the secretion of the fusion protein. Methods The c DNA of humanIFN-γ and GM-CSF was obtained by RT-PCR respectively. And the obtained fragments were amplified by PCR and thenlinked by（Gly4Ser）3to produce a fusion gene of GMI- γ. The fusion gene was amplified first and then inserted into thedownstream of promoter HSP60 of E.coli-BCG shuttle-vector p MV261 to construct a secreting expression plasmid p MVGMI-γ. Then the p MVGMI-γ was transformed into competent cells of E.coli DH5 alpha,and the positive clones were selected onkanamycin resistant LB culture medium plate. The positive plasmids were extracted and transformed into competent cells ofBCG by electric shock. The target protein was purified by precipitation of（NH4）2SO4and molecular chromatography columnBio-Gel P-6 DG. The purification results were identified by 10% SDS-PAGE and Western blotting for the expression of fusiongene GMI- γ in r BCG. Results The RT- PCR products of target genes GM- CSF and IFN- γ were 461 and 432 bp,respectively,which were in agreement with the expected values. The fusion gene GMI-γ（462＋435-45=852 bp） were correctlyinserted into the plasmid p MV261 and expressed in BCG,after the identification by double restriction endonuclease digestion,PCR amplification and gene sequencing. Conclusion The plasmid p MVGMI- γ is constructed successfully and cansecretively express GMI-γ in BCG. This obtain plasmid found a basis for the immune activity of r BCG and the development ofnew Mycobacterium tuberculosis vaccine.