TONG Yi-Gang, DU Yong, XU Jing, LI Jing-Yun, LU Yan-Xi, BAO Zuo-Xi and WANG Hai-Chao. High Level Soluble Expressiion,One-Step Purification and Characterization of HIV-1 p24 Protein[J]. Virologica Sinica, 2000, 15(2): 116-121.
Citation: TONG Yi-Gang, DU Yong, XU Jing, LI Jing-Yun, LU Yan-Xi, BAO Zuo-Xi, WANG Hai-Chao. High Level Soluble Expressiion,One-Step Purification and Characterization of HIV-1 p24 Protein .VIROLOGICA SINICA, 2000, 15(2) : 116-121.

HIV-1p24蛋白在大肠肝菌中的高效可溶性表达、一步纯化及抗原性分析

  • 合成引物扩增HIV1p24基因,并将其克隆到pQE30质粒中,使其在大肠杆菌E.coliM15中以IPTG诱导高效表达,经SDSPAGE分析,该表达产物约占菌体总蛋白20%,并且以可溶蛋白的形式存在于细菌裂解液上清之中。经镍离子柱亲和层析一步纯化,洗脱产物中p24蛋白纯度达95%。ELISA分析表明,该蛋白可与HIV感染者血清发生特异性免疫反应。以此蛋白交联Sepharose4B,亲和层析纯化HIV感染者血清中的抗体,用所得抗体与HIV确认试剂反应,发现该纯化抗体仅与确认试剂中的p24蛋白反应。上述结果表明在大肠杆菌中已经高效表达了可溶性HIV1p24蛋白,该蛋白具有良好的抗原性。

High Level Soluble Expressiion,One-Step Purification and Characterization of HIV-1 p24 Protein

  • The HIV 1 p24 gene (gag gene) was amplified by polymerase chain reaction (PCR) and cloned into an E.coli expression vehicle pQE30 between BamH I and Kpn I sites. After induction with IPTG, the transformed E.coli strain M15 expressed the HIV 1 p24 gene efficiently. The recombinant p24 protein was expressed as a soluble protein, composing 20% of the total protein. After onestep purification with Ni NTA + affinity chromatography, the protein was purified to almost homogeneity. When applied to ELISA, the p24 protein exhibited good specific reactivity with sera of HIV infected individuals. The antibodies against recombinant p24 protein from HIV infected plasma were purified and confirmed with HIV Western blot kit that the purified antibodies can only react with HIV 1 p24 antigen. It suggests that the recombinant HIV 1 p24 protein is suitable for assembling the HIV diagnostic kits.

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    High Level Soluble Expressiion,One-Step Purification and Characterization of HIV-1 p24 Protein

    • 1. Institute ofMicrobiology and Egidemiology,Beijing 100071,China

    Abstract: The HIV 1 p24 gene (gag gene) was amplified by polymerase chain reaction (PCR) and cloned into an E.coli expression vehicle pQE30 between BamH I and Kpn I sites. After induction with IPTG, the transformed E.coli strain M15 expressed the HIV 1 p24 gene efficiently. The recombinant p24 protein was expressed as a soluble protein, composing 20% of the total protein. After onestep purification with Ni NTA + affinity chromatography, the protein was purified to almost homogeneity. When applied to ELISA, the p24 protein exhibited good specific reactivity with sera of HIV infected individuals. The antibodies against recombinant p24 protein from HIV infected plasma were purified and confirmed with HIV Western blot kit that the purified antibodies can only react with HIV 1 p24 antigen. It suggests that the recombinant HIV 1 p24 protein is suitable for assembling the HIV diagnostic kits.

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