分泌表达Der p2的重组BCG的构建与鉴定

Construction and identification of the recombinant BCG expressing Der p2 in form of secreting protein

目的构建并鉴定能够分泌表达外源蛋白——屋尘螨抗原(Derp2)的基因重组卡介苗(rBCG)。方法采用PCR技术,从结核分枝杆菌H37Rv基因中扩增信号肽Alpha抗原(α-ss)的穿膜区基因,经测序鉴定后克隆入胞内表达Derp2的rBCG中。通过Westernblot鉴定目标抗原在rBCG中的表达。结果经测序证实,所克隆的α-ss信号肽基因序列正确,构建的Derp2-rBCG能完成在大肠埃希菌(E.coli)和牡牛分枝杆菌细胞之间的穿梭,并介导抗生素抗性基因表达。外源基因Derp2表达于BCG培养上清。结论成功构建了以分泌方式表达外源蛋白的Derp2-rBCG。

Objective To construct and identify the recombinant BCG （bacille Calmette-Guerin） expressing exogenous antigen, namely Dermatophagoides pteronyssinus （house dust mite）, major allergen （Der p2） in form of secreting protein. Methods The gene fragments containing a-ss signal peptide gene were amplified by polymerase chain reaction （PCR） from the Mycobacteria tuberculosis H37Rv and cloned into the Der p2-rBCG, which can express Der p2 in intracellular pattern. Then the Der p2 gene was expressed by the rBCG in form of secreting protein, and it was identified by Westem blotting. Results The sequence of signal peptide gene cvss was verified by sequencing identification. The constructed Der p2-rBCG could shuttle from E. coli to Mycobacteria to mediat the expression of antibiotic resistance gene and it served as a vector to express the Der p2 gene as a secreted protein. Conclusions The Der p2-rBCG, which can express exogenous antigen gene in form of secreting protein, has been constructed successfully.