摘要
目的:为了方便实验室工作中HIV-1B'/C亚型及C亚型Tat蛋白的检测,制备了相应的Tat蛋白及其抗体。方法:将我国HIV-1B'/C亚型流行株tat基因的第1个外显子和HIV-1C亚型tat基因的第2个外显子融合在一起,将密码子替换为大肠杆菌的优势密码子,通过合成引物、PCR拼接的方法,获得目的基因序列;在原核系统中与pET32a+载体中的His·Tag、Trx·Tag及S·Tag进行融合表达;目的蛋白经Ni+金属螯合层析柱纯化后,用于免疫家兔,制备多克隆抗体。结果:PCR拼接获得306bp的目的基因序列;在原核系统中融合表达得到相对分子质量约31000的融合蛋白,占菌体总蛋白的21%。纯化后的融合蛋白免疫家兔,制备了多克隆抗体,Western印迹结果显示,获得的多克隆抗体与HIV-1B'/C亚型的Tat蛋白反应良好;间接免疫荧光结果表明,获得的多克隆抗体与HIV-1B'/C亚型和C亚型的Tat蛋白都能产生特异性反应。结论:制备的多克隆抗体能够使用间接免疫荧光方法检测HIV-1C亚型的Tat蛋白,使用Western印迹方法和间接免疫荧光方法都能检测HIV-1B'/C亚型的Tat蛋白。
Objective： In order to detect the Tat protein of HIV-1 B＇/C subtype and C subtype conveniently in lab work, the corresponding Tat protein and its antibody were prepared. Methods： The first extron of tat gene of HIV-1 B＇/C subtype and the second extron of tat gene of HIV-1 C subtype were fused as target gene, and the codons of target gene were replaced with the preferred codons of E.coli through primers mutation and PCR splicing. The optimized target gene was fused again with the gene of His-Tag, Trx-Tag and S·Tag in pET32a＋ vector. Then, the fusion protein was expressed in prokaryotic system. The Tat fusion proteins were purified by nickel-chelating chromatography. The purified Tat fusion protein was used to immune rabbit to prepare polyclonal antibody. Results： The 306 bp aim gene was gained through PCR splicing, and the approximate 31 kD fusion protein was expressed efficiently in prokaryotic system. The ratio of Tat fusion protein to total bacteria proteins was 21%. The purified fusion protein was used to prepare polyclonal antibody. The polyclonal antibody could react well only with Tat protein of HIV-1 B＇/C subtype demonstrated by Western-blot, but could react efficiently with both Tat protein of HIV-1 B＇/C subtype and C subtype showed by indirect immunofluorescence. Conclusion： The polyclonal antibody can be used to detect Tat protein of HIV-1 B＇/C subtype by Western-blot, and it can be used to detect Tat protein of HIV-1 B＇/C subtype and C subtype by indirect immunofluorescence.
出处
《生物技术通讯》
CAS
2006年第2期142-145,共4页
Letters in Biotechnology
基金
中国综合性国际艾滋病研究项目(CIPRA)(1U19A15191501)
国家高技术研究发展计划项目(2003AA219080)
