表达呼吸道合胞病毒f212-489蛋白乳酸乳球菌的构建.docVIP

组成型表达人呼吸道合胞病毒F212-489蛋白乳酸乳球菌的构建 冯金1，洪愉2，毛普加1，黄芬1，井申荣1，曾韦锟1 （650500云南，昆明理工大学生命科学与技术学院 1，650032云南，成都军区昆明总医院核医学科2） [摘要] 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，通过荧光显微镜观察，SDS和免疫印记方法检测目的蛋白。结果 在重组菌中检测到EGFP发出的荧光，Western-blot检测到f212-489在乳酸乳球菌中的表达。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。 [关键词] 人类呼吸道合胞病毒，F蛋白，乳酸乳球菌 [中图分类号] R37 Constitutive expression of human respiratory syncytial virus protein F212-489 in Lactococcus lactis Feng Jin1, Hong Yu2, Mao Pujia1,Huang Fen1, Jing Shenrong1, Zeng Weikun1 (1Faculty of Life Science and Biotechnology, Kunming University of Science and Technology, Kunming, 650500, Yunnan,China,2 Department of nuclear medicine, Kunming General Hospital of Chengdu Military Command ,Kunming,650032,Yunnan,China.) [Abstract] Objective To construct a Lactococcus lactis system which could express the truncated F1 protein（F212-489） of human respiratory syncytial virus. To identify the immunoreactivity and then evaluate the possibility of recombinant bacteria as an oral vaccine. Methods Firstly, egfp and f212-489 gene was amplied by PCR and inserted into the pMD18T vector for sequencing. Secondly, Recombinant bacteria pMG36e-f212-489/MG1363 was constructed. Finally, SDSand Western-blot were used to investigate the expressing protein. Results Fluorescent of EGFP were found from recombinant bacteria. F212-489 was detected in Lactococcus lactis with Western-blot. Conclusions The protein f212-489 could be correctly expressed by the recombinant Lactococcus lactis and kept its immunoreactivity. [Keywords] Human Respiratory Syncytial Virus; Fusion protein; Lactococcus Lactis Supported by the General Program of ?National Natural Science Foundation of China. Corresponding author: Zeng Weikun, E-mail: zengweikun@ 呼吸道合胞病毒（RSV）是婴幼儿下呼吸道感染最的病原体，免疫缺陷病人及老年人也是RSV 的易感人[1,2]，其临床症状以低热和下呼吸道疾病为主，婴幼儿多出现咳嗽和憋喘，严重者可致死亡[3]。在RSV疫苗研究中发现，疫苗免疫机体时，常会使机体出现Th2型的免疫极化，进而引发免疫病理反应[4]。