摘要
目的制备具有生物活性的HIV-1 env蛋白,利用量子点的特殊光学性能结合免疫分析技术,建立快速、简便的免疫凝集分析检测HIV-1gp41抗体方法。方法以HIV-1 env基因质粒为模板,采用体外快速翻译系统(RTS)线性模板试剂盒制备线性模板,用RTS100试剂盒合成env蛋白,用磁珠法纯化蛋白,用Western Blot方法鉴定蛋白活性。纯化后的HIV-1 env蛋白采用量子点标记,与样品液中的抗HIV-1gp41抗体进行免疫反应,根据量子点的荧光信号的变化,定量检测样品液中抗gp41抗体的浓度。结果体外快速翻译系统表达出相对分子量约为97KD的env蛋白;Western blotting证实纯化后的蛋白具有生物活性,量子点标记的env蛋白与山羊抗HIV-1gp41抗体发生了免疫凝集反应,使量子点发生荧光淬灭,荧光信号强度与抗体浓度呈负相关。结论HIV-1 env蛋白可采用RTS系统进行大量表达,分离纯化后的env蛋白具有生物学活性,建立的量子点标记的免疫凝集反应方法检测HIV gp41结果准确,方法简便。
[Objectives] To prepare HIV-1 env protein, and use quantum dots to label HIV-1 env protein, and establish the rapid immunoassay method for HIV-1 gp41 antibody. [Methods] The env gene fragments were obtained from the plasmid with HIV-1 env by Polymerase Chain Reaction, and were cloned into T vector, and then were sequenced. The linear env gene fragments were prepared by Rapid Translation System Linear Template Kit, the env proteins were obtained by Rapid Translation System Kit, and were purified by magnetic bead method. The env protein＂ s bioaetivity was identified by Western Blotting. The prepared env proteins were labeled with CdTe quantum dots, the goat anti-HIV-1 gp41 IgG antibodies were detected by CdTe quantum dots-labeled env proteins, the fluorescent signals were recorded by fluorescent spectroscopy. [Results] The env gene was cloned into T vector, its gene sequence was right. The env proteins were successfully expressed by Rapid Translation System, and were purlfled by magnetic bead method, Western Blotting confirmed that the prepared env proteins owned bioactivity. The env proteins were successfully labeled with CdTe quantum dots. The CdTe quantum dots labeled env proteins could react with anti-HIV-1 gp41 IgG antibodies, and formed the env-gp41-CdTe quantum dots complex, the fluorescent intensity of post-reaction products were negatively associated with the concentration of anti-HIV-1 gp41 IgG antibodies in sample. [Conclusion] The HIV-1 env proteins were successfully prepared, the purified proteins had bioactivity, the fluorescent immunoassays method to detect HIV-1 gp41 IgG antibodies in sample with CdTe quantum dots labeled env proteins was established.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2009年第10期1474-1478,共5页
China Journal of Modern Medicine
基金
国家973计划(No:2005CB723400-G)
国家863重点项目(No:2007AA022004)
国家自然科学基金(No:30771075
No:30672147)
上海市科委纳米专项基金(No:0752nm024)
上海市科委基金(No:072112006-6)资助
