摘要
目的:为研究HIV-1 NCp15的生物学活性,制备HIV-1 NCp15蛋白及其鸡卵黄抗体。方法:用PCR的方法扩增编码NCp15基因序列,将其克隆到原核表达载体PGEX4T1中表达HIV-1 NCp15蛋白,用HIV阳性血清做Western blot鉴定目的蛋白。以纯化的NCp15蛋白抗原免疫产蛋鸡,制备卵黄抗体。通过酶联免疫吸附实验(ELISA),Western blot检测抗体滴度及特异性。结果:成功构建原核表达载体PGEX4T1-NCp15,并可在大肠杆菌BL21(DE3)中诱导表达,得到相对分子质量约45ka的NCp15融合蛋白,经Western blot鉴定正确。纯化蛋白免疫产蛋鸡,制备的鸡卵黄抗体IgY具有较强免疫特异性。结论:得到纯化的HIV-1NCp15蛋白,制备的IgY能够检测HIV-1病毒蛋白NCp15,为进一步研究HIV-1奠定了实验基础。
Objective： To prepare HIV-1 nucleocapsid（NC） protein（NCp15） and its antibody IgY for studying the biological activity of HIV-1 NCp15. Methods： The full length gene fragment of NCp15 was amplified by PCR, and then cloned into PGEX4T1 prokaryotic expressing vector. The recombinant protein NCp15-GST was induced by IPTG in E. coil BL21 and the expressed protein was detected by Western blot assay. The purified protein was used to immune chicken to prepare egg yolk immunoglubin（IgY）, and the specificity was identified by Western blot assay. Results： The plasmid PGEX4T1-NCp15 was successfully constructed and HIV-1 NCp15 protein could be expressed in E. coli BL21 with high efficiency. The recombinant protein was characterized with HIV-1 positive plasma by Western blot. Chicken immunized with the purified protein produced high titer IgY. Conclusion： Purified protein HIV-1 NC p15 with high antigenicity was obtained,and the prepared IgY could be used to detect the HIV-1 viral protein, which provided reliable tools for the future HIV-1 research.
出处
《食品科技》
CAS
北大核心
2009年第3期28-32,共5页
Food Science and Technology
基金
2007湖北省卫生厅科研基金项目(JX3B80)
