摘要
目的基于乳酸乳球菌和鼠疫耶尔森菌F1抗原,制备鼠疫拟菌颗粒疫苗。方法基于λ-Red一步重组技术,构建BL-21大肠杆菌htr A基因缺失突变株;将鼠疫菌F1抗原基因和PA基因克隆至p ET-28a(+)质粒中构建F1-PA融合蛋白表达载体,将此载体分别导入到BL-21大肠杆菌野生株和其htr A基因缺失突变株中构建F1-PA融合蛋白表达菌株; SDS-PAGE电泳分析F1-PA融合蛋白表达;镍柱亲和层析纯化包涵体蛋白,梯度稀释透析法复性F1-PA融合蛋白;乳酸乳球菌经热酸处理制备拟菌颗粒;革兰染色和透射电镜分析拟菌颗粒的形态;拟菌颗粒分别与F1-PA融合蛋白或F1蛋白共孵育制备拟菌颗粒疫苗。结果 F1-PA融合蛋白在大肠杆菌野生株和htr A缺失突变株中均以包涵体形式表达。拟菌颗粒对F1-Pa融合蛋白的吸附能力明显高于对F1蛋白的吸附能力。F1-PA融合蛋白在大肠杆菌htr A突变株中的表达量明显高于在野生株中的表达量。结论大肠杆菌htr A突变株用于表达F1-PA融合蛋白优于野生株。借助于肽聚糖锚钩蛋白,可将鼠疫菌F1抗原结合到乳酸乳球菌拟菌颗粒上制备鼠疫拟菌颗粒疫苗。
Objective To prepare a bacteria-like particle vaccine against plague based on Lactococcus lactis and F1 antigen of Yersinia pestis. Methods The construction of htr A-deficient mutant of E. coli BL-21 was based on λ-Red onestep recombination technology. The F1 antigen and PA genes were cloned into p ET-28 a( +) to construct the expression vector of F1-PA fusion protein. The expression vector was respectively transferred into E. coli BL-21 and htr A-deficient mutant of E. coli BL-21 to construct the expression strains that could yield F1-PA fusion protein. The expressed F1-PA fusion protein was detected by SDS-PAGE electrophoresis. The inclusion body protein was purified by Ni2 +affinity column chromatography,and then refolded by gradient dialysis. Bacteria-like particles were prepared by hot acid treatment,and their appearance was observed by Gram staining and transmission electron microscopy. The bacteria-like particle vaccine was prepared incubating the bacteria-like particles with F1-PA fusion protein or F1 protein. Results The F1-PA fusion protein was accumulated as an inclusion body in wild E. coli BL-21 or htr A-deficient mutant of E. coli BL-21. The bacterialike particles showed a higher adsorption capacity for F1-PA fusion protein than F1 protein. The expression level of F1-PA fusion protein in htr A-deficient mutant of E. coli BL-21 was higher than that in E. coli BL-21. Conclusion The htr Adeficient mutant of E. coli BL-21 has an advantage over E. coli BL-21 in F1-PA fusion protein preparation. Y. pestis F1 antigen can be easily bound to the bacteria-like particles to a prepare plague vaccine by means of peptidoglycan anchor protein.
作者
李璐
刘万兵
周亚洲
纪宇欣
周冬生
杨瑞馥
王效义
LI Lu;LIU Wan-bing;ZHOU Ya-zhou;JI Yu-xin;ZHOU Dong-sheng;YANG Rui-fu;YANG Xiao-yi(Anhui Medical University,Hefei 230032 Anhui,China;State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071, China)
出处
《军事医学》
CAS
CSCD
北大核心
2018年第7期539-544,共6页
Military Medical Sciences
基金
国家重点研发计划资助项目(2016YFC1202600).
