摘要
目的纯化铜绿假单胞菌(Pseudomonas aeruginosa)溶血素共调节蛋白(Hemolysin co-regulated protein,Hcp)并制备抗血清。方法从NCBI网站查到铜绿假单胞菌Hcp蛋白序列,采用Signal IP 4.1预测定位及信号肽情况;登录Swiss model网站预测三级结构情况;登录www.iedb.org网站预测其B细胞表位。根据Hcp基因序列设计特异性引物,以铜绿假单胞菌基因组DNA为模板,PCR扩增Hcp基因,经双酶切后连到pET28a。将重组质粒pET28a-Hcp转化大肠埃希菌BL21(DE3),然后用IPTG诱导重组Hcp蛋白表达。使用镍柱亲和层析法纯化目的蛋白。用Hcp蛋白免疫BALB/c小鼠,ELISA法检测抗血清效价。结果 Hcp定位在细菌细胞浆,无信号肽,6个单体蛋白组成一个环状的中空结构,含有多个B细胞表位。构建的表达质粒pET28a-Hcp能表达23.9×10~3大小的Hcp蛋白,镍柱层析后得到高纯度的Hcp蛋白,重组Hcp蛋白诱导的抗体效价大于1∶10 240。结论成功重组表达铜绿假单胞菌Hcp蛋白及高滴度的抗血清,为研究Hcp的功能奠定了实验基础。
Objectives To purify the hemolysin co-regulated protein(Hcp) of Pseudomonas aeruginosa from E. coli and to prepare antiserum against the Hcp protein. Methods Amino acids of the protein sequence of Hcp from P. aeruginosa were determined from NCBI, and their localization and the presence of signal peptides were predicted using Signal IP 4.1, the protein’s tertiary structure was predicted using the Swiss model website, and its B-cell epitopes were predicted using the website www.iedb.org. Specific primers were designed based on the Hcp gene sequence, and the Hcp gene was amplified using PCR. The Hcp gene was ligated to a pET28 a plasmid after double digestion. The recombinant plasmid pET28 a-Hcp was transformed into E. coli BL21(DE3), and then expression of the recombinant Hcp protein was induced with IPTG. The protein of interest was purified using nickel column affinity chromatography. The Hcp protein was used to immunize BALB/c mice to produce antiserum, and the titer of antiserum was measured using ELISA. Results The Hcp protein is located in the bacterial cytoplasm, it lacks signal peptides, and it contains multiple B-cell epitopes. Six monomeric Hcp proteins formed a circular hollow structure. The Hcp gene, 519 bp in length, was obtained using PCR. The expression plasmid pET28 a-Hcp was successfully constructed, the recombinant 23.9×10~3 Hcp protein was expressed, and a highly pure Hcp protein was obtained after purification. The antibody titers in sera of mice administered the recombinant Hcp protein were above 1:10240. Conclusion The Hcp of P. aeruginosa and a high-titer antiserum were successfully produced, laying the experimental basis for study of the function of Hcp.
作者
申文娟
张凯
肖宏
李冉辉
SHEN Wen-juan;ZHANG Kai;XIAO Hong;LI Ran-hui(Intensive Care Unit,Second Affiliated Hospital of the University of South China,Hunan,China 421001;Institute of Pathogen Biology,University of South China,Hengyang)
出处
《中国病原生物学杂志》
CSCD
北大核心
2019年第10期1130-1134,共5页
Journal of Pathogen Biology
基金
湖南卫计委课题项目(No.B2017060)
关键词
铜绿假单胞菌
溶血素共调节蛋白
蛋白纯化
抗血清
Pseudomonas aeruginosa
hemolysin co-regulated protein
protein purification
antiserum
