组成型表达人呼吸道合胞病毒F_(212-489)蛋白乳酸乳球菌的构建

Constitutive expression of human respiratory syncytial virus protein F_(212-489) in Lactococcus lactis

目的构建能表达呼吸道合胞病毒截短F1蛋白(F212-489)的乳酸乳球菌,并检测表达蛋白的免疫反应性,以评估此重组菌作为口服活菌疫苗的可行性。方法 PCR扩增绿色荧光基因egfp以及截短f1基因(f212-489),经TA克隆测序后,将目的片段构建至pMG36e载体,电转化至乳酸乳球菌中鉴定并表达,通过荧光显微镜检测绿色荧光蛋白EGFP的表达,通过SDS-PAGE和Western blot检测F212-489蛋白的表达。结果荧光显微镜观察到重组菌pMG36e-egfp/MG1363有绿色荧光表达,Western blot检测到重组菌pMG36e-f212-489/MG1363中F212-489蛋白的表达。结论构建的乳酸乳球菌能够组成型表达F212-489蛋白,其蛋白具备免疫反应性。

Objective To construct a Lactococcus lactis system which could express the truncated F1 protein （F212-489） of human respiratory syncytial virus and to identify the immunoreactivity of the obtained protein in order to evaluate the possibility of recombinant bacteria as an oral vaccine. Methods Firstly, egfp and f212-489 gene were amplified by PCR, and then the product was inserted into the pMD18T vector for sequencing. Secondly, recombinant bacteria pMG36e-egfp/MG1363 and pMG36e-f212-489/MG1363 were constructed. Finally, fluorescence microscopy, SDS-PAGE and Western blotting were used to investigate the expressed protein. Results Expression of enhanced green fluorescent protein （EGFP） was found in the recombinant bacteria pMG36e-egfp/MG1363. F212-489 was detected in the recombinant bacteria by Western blotting. Conclusion The protein F212-489 is correctly expressed by the recombinant Lactococcus lactis with its immunoreactivity.