【摘要】 目的构建铜绿假单胞菌(Pseudomonas aeruginosa,PA)XN-1的全基因组文库,筛选铜绿假单胞菌新的疫苗候选抗原。方法提取我国分离的临床PA XN-1菌株全基因组,Sau3AⅠ酶切成随机片段后经连接至Bam HⅠ酶切后的pMal-c5x载体,转化至X-blue获得随机重组子。随机重组子经IPTG诱导后使用溶菌酶裂解,收集含有融合的麦芽糖结合蛋白(MBP)的随机重组子的裂解上清,制成含有PA全基因组的蛋白随机文库。通过ELISA方法分别检测单个随机文库蛋白的免疫反应性。将经典的PA强反应抗原PcrV做为参照,分别计算单个随机抗原的相对反应强度并进行比较。提取反应较强的重组子的质粒,测定DNA序列后进行翻译和比对,得到整个强反应性抗原的氨基酸信息。结果成功构建PA XN-1的全基因组文库,筛选到强反应性抗原13个,其中4个抗原的反应强度超过PcrV,分别为Peptidyl-prolyl cis-trans isomerase D、Phosphoenolpyruvate-protein phosphotransferase(PtsP)、Phosphate-starvation-inducible E和Very short patch repair endonuclease。结论基于高效表达系统的PA全基因组文库构建成功,可用于PA及其它病原的疫苗候选抗原的筛选研究。更多还原

【Abstract】 This study was designed to build up genome-wide antigen screening library of Pseudomonas aeruginosa(PA) and select their new antigens. The whole genome of PA XN-1 was extracted and digested into random fragments by Sau3 A Ⅰ, which were then ligated into pMal-c5 x vector after treatment with Bam HⅠ, and transformed into X-blue for selection of random recombinants. Random recombinants were induced by IPTG and lysed by lysozyme. Lysis supernatants which contains MBP(maltose binding protein) fusion protein were collected,thus random library of recombinants from the whole genome of PA XN-1 was constructed. Enzyme linked immunosorbent assay(ELISA) was employed to measure the immune-reactivity of each recombinant from the random library. The previously reported strong reaction antigen PcrV was used as a positive reference,and the relative strength of each recombinant was calculated and analyzed. The DNA of the highlyreactive recombinants was sequenced and its amino acid information was obtained by BLAST search. Fifteen strong reaction antigens were screened, 4 of which showed higher immune response than PcrV, namely Peptidyl-prolyl cistrans isomerase D, Phosphoenolpyruvate-protein phosphotransferase(PtsP), Phosphate-starvation-inducible E, and Very short patch repair endonuclease. In conclusion, the screening library of genome-wide Pseudomonas aeruginosa antigens has successfully constructed, which could be used for further study of vaccine development against Pseudomonas aeruginosa and other pathogens.更多还原