摘要
目的:构建基于中国HIV-1 CRF01_AE重组亚型包膜糖蛋白gp41 NHR结构域N51的亚单位疫苗,并进行免疫原性研究。方法:设计4条引物,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,构建重组质粒pFUSE/N51Fd并进行序列测定。Western blot法检测N51FdFc-AE重组蛋白的表达。用纯化蛋白免疫BALB/c小鼠后,ELISA法检测小鼠的抗体反应。结果:成功构建了pFUSE/N51Fd重组质粒,N51FdFc-AE重组蛋白在真核体系获得了高效表达,Western blot结果显示在相对分子质量(Mr)35 000处有目的蛋白条带。小鼠抗血清能特异性识别源于gp41 NHR的抗原,效价高达1∶102 400,平均效价为1∶51 200。结论:改造后的亚单位疫苗能有效激活机体的免疫响应,可用于HIV候选疫苗的研发。
AIM： To design and construct an HIV-1 subunit vaccine containing the N-terminal heptad repeat （NHR） domain N51 in gp41 of the HIV-1 CRF01_AE recombinant subtype in China and study its immunogenicity. METHODS： Two pairs of primers were designed to amplify DNA fragment encoding N51Fd gene, which was then subcloned into pFUSE-higG1-Fc2 vector. The recombinant plasmid pFUSE/N51Fd was confirmed by DNA sequencing. Western blotting was used to measure the correct expression of the recombinant protein N51FdFc-AE. The N51FdFc- AE protein was used to immunize BALB/c mice, and the specific antibody response was measured by ELISA. RESULTS： A recombinant plasmid pFUSE/N51Fd was successfully constructed and the N51FdFc-AE recombinant protein was expressed effectively in 293T cells. The purified N5IFdFc-AE recombinant protein （35 kD） was detected by Western blotting with rabbit anti-gp41 N/C peptide antibodies. The mouse anti-sera could specifically recognize the antigens derived from gp41 NHR. The titers of the specific antibodies were up to I：102 400 with an average titer of 1：51 200. CONCLUSION： The recombinant N51FdFc-AE protein can effectively induce the gp41 NHR-specific immune responses in BALB/c mice and could be used as an immunogen for design of HIV-1 subunit vaccine.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第12期1250-1253,1257,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金-广东省联合基金重点项目(U0832001)
