摘要
目的:探讨M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域表达融合蛋白的可行性。方法:以纯化的HIV-1感染者血清多克隆抗体为配体,在噬菌体展示随机十二肽库中进行生物淘洗,经ELISA鉴定阳性克隆,DNA测序,确定优势表位。将优势表位及两个优势表位的串联体分别与M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域连接,克隆入PQE30载体进行蛋白表达,再以表达蛋白为抗原检测HIV-1感染者血清中的抗体。结果:成功筛选到位于HIV-1 gp41蛋白上的3组优势抗原表位(HGPKDAETTAIW;AAFKDNQLLRIW;AAFKDNQLTRIW),3组优势表位及表位串联体(YGPKDAETTAIW-GGGS-SCSAKFTCTTQI)在PQE30载体中实现可溶性融合表达。重组蛋白具有良好的抗原性,能与不同的HIV-1抗体阳性血清呈特异反应。结论:用M13噬菌体PⅢ蛋白Ⅰ-Ⅱ结构域表达HIV-1 gp41抗原表位是可行的。
Objective： To explore the feasibility of expressing a fusion protein with Ⅰ - Ⅱ domain of M13 phage PⅢ protein. Methods： The polyelonal antibodies specific to HIV- 1 gp41 protein were prepared from HIV - 1 B＇ IgG positive plasma by affinity chromatography 4B coupled with HIV- 1 gp41 protein. The phage display 12 peptides libraries were biopanned first with the antibody, and then non- specific phages were subtracted by purified IgG frown non- HIV sera. After three round screening, the positive phages were tested by ELISA for their reactivity with anti- HIV IgG antibodies, and their major display peptides were determined by DNA sequencing. The major epitopes and two epitopes series were ligated with Ⅰ - Ⅱ domain of M13 phage PⅢ protein, respectively. The epitopes were expressed in PQE30 vector. Results： Three major HIV - 1 gp41 epitopes （YGPKDAETTAIW; AAFKDNQLLRIW; AAFKDNQLTRIW） were determined. The epitopes and two epitopes series （YGPKDAKTTAIW- GGGS - SCSAKFTCTTQI） were expressed as fusion protein in PQE30 vector. Recombinant proteins showed an actual positive reactivity with HIV- 1 IgG antibody from different sera, Conclusion： The design of expressing HIV- 1 gp41 epitopes gene with Ⅰ -Ⅱ domain of M13 phage PⅢ protein is practical.
出处
《现代预防医学》
CAS
北大核心
2006年第12期2313-2315,2319,共4页
Modern Preventive Medicine
