摘要
目的对来源于HIV-1中国流行株CRF07_BC的包膜糖蛋白gp41 NHR结构域的N51进行表达和结构及抗原性分析。方法运用重叠延伸PCR方法扩增出N51Fd基因,将其插入真核表达载体pFUSE-hIgG1-Fc2,并进行核苷酸序列测定。利用生物信息学软件、圆二色谱法、免疫印迹法对表达的N51FdFc-BC重组蛋白进行结构和抗原性分析。结果成功构建pFUSE/N51Fd-BC表达载体,并在真核表达体系实现了目的蛋白的高效表达。免疫印迹结果显示该重组蛋白大小约为35 000,可与抗HIV-1 gp41 N/C多肽的抗体反应。生物信息学分析显示N51FdFc-BC重组蛋白相对分子质量为34 315.1,等电点PI为7.59,且形成了无规则卷曲结构,易于与抗体反应,可作为抗原。圆二色谱的分析与生物信息学软件预测的结果一致。结论N51FdFc-BC重组蛋白具有无规则卷曲结构,可适合作为HIV-1亚单位疫苗的免疫原。
Objective To express N51 derived from the N-terminal heptad repeat （NHR） domain in gp41 of the HIV-1 CRF07_BC strain and analyze its molecular structure and antigenicity. Methods Overlapping PCR was used to amplify the DNA fragment encoding N51Fd gene, which was then subcloned into the vector pFUSE-hIgG1-Fc2. The construct was confirmed by DNA sequencing. The structure and antigenicity of the recombinant protein N51FdFc-BC were analyzed using bioinformatic software, circular dichroism, and Western blotting. Results A recombinant expression vector pFUSE/N51Fd-BC was successfully constructed. N51FdFc-BC recombinant protein with a relative molecular mass of about 35 000 was effectively expressed in mammalian 293T cells and could be recognized by rabbit antibodies against HW-1 gp41 N/C peptides as shown by Western blotting. Bioinformatic analysis showed that the recombinant protein N51FdFc-BC, with a relative molecular mass of 34 315.1 and a PI of 7.59, formed a secondary structure of random coil to allow its interactions as an antigen with antibodies. Circular dichroism measurement confirmed the random coil structure of N51FdFc-BC protein. Conclusion The recombinant protein N51FdFc-BC has a random coil structure and can be used as an immunogen for development of HIV-1 subunit vaccine.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2012年第12期1737-1740,共4页
Journal of Southern Medical University
基金
国家自然科学基金-广东省联合基金重点项目(U0832001)~~
