家畜生态学报2012，Vol.33Issue(2)：31-36,6.
H5N1亚型禽流感病毒RNA聚合酶PB1_P亚基片段的表达、纯化和结晶
Expression, Purification and Preliminary Crystallization of Avian Influenza Virus (H5N1) RNA Polymerase PB1_P Subunit
范克伟 1宠海 2陈吉龙 3黄志勇 4吴德峰4
作者信息
- 1. 福建农林大学生命科学学院,福建福州350002
- 2. 清华大学医学院,北京100084
- 3. 中国科学院微生物研究所,北京100101
- 4. 福建农林大学动物科学学院,福建福州350002
- 折叠
摘要
Abstract
This research was conducted to clone and express the PB1_P protein of avian influenza virus H5N1 RNA polymerase and purify PB1_P recombinant protein with high concentration and its preliminary crystals,which could be useful for the further study of the function and structure of the influenza virus polymerase and the development of a new diagnostic kit. According to the sequence of polymerase basic protein 1 PB1 gene in GenBank.a pair of specific primers were designed. Polymerase domain (PB1_P) of avian influenza virus PB1 gene was amplified by RT-PCR,and inserted into pET-28a,then the constructed recombinant plasmid was analyzed. Then, the positive pET-28a/PBl_P plasmids were transformed into E. coli BL2KDE3)competent cell and induced for 4 h, at 37 ℃ by 0. 5 mmol/L IPTG. The recombinant fusion protein PB1_P was successfully expressed and identified by SDS-PAGE and Western-blot and then purified by Ni-NTA His Bind Resins and column chromatography. After purification, the preliminary crystals of PB1_P recombinant protein was obtained by the hanging drop vapor diffusion method. The pET-28a/PBl_ P recombinant plasmid was successfully reconstructed. Analysis of SDS-PAGE showed that the fusion protein PB1_P was successfully cloned and expressed. The result of Western-blot indicated that the recombinant had specially immunologic reactionogenicty with postive serum. The preliminary crystals of PB1_P recombinant protein was obtained after purification and crystallization screening. This study provides a sound basis for further study on biological function and of PB1 and the development of a new avian influenza virus H5N1 detection kit.关键词
流感病毒H5N1/PB1_P/克隆/表达纯化/蛋白结晶Key words
Avian influenza virus H5N1/ PB1_P/ clone/expression and purification/ protein crystalli-zation分类
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范克伟,宠海,陈吉龙,黄志勇,吴德峰..H5N1亚型禽流感病毒RNA聚合酶PB1_P亚基片段的表达、纯化和结晶[J].家畜生态学报,2012,33(2):31-36,6.
