摘要
旨在对牛副流感病毒3型(BPIV3)NP基因进行分段克隆及原核表达,获得纯化蛋白。根据GenBank中公布的牛副流感病毒3型NP蛋白基因序列(JQ063064)设计合成2对特异性引物,采用反转录-聚合酶链反应(RT-PCR)的方法,以牛胚肾细胞(MDBK)培养的BPIV3NM09株病毒液提取的RNA为模板,扩增获得2段NP基因序列(NP1、NP2),将2个片段克隆到PGEX-6P-1表达载体,构建具GST标签的重组质粒PGEX-NP1、PGEX-NP2,将2种阳性重组质粒分别转化至大肠埃希菌BL21(DE3),优化诱导表达条件进行可溶性分析,并用SDS-PAGE及Western blot进行鉴定,同时利用亲和层析法将重组蛋白纯化。最终获得大小分别为42.3ku、38.5ku的重组蛋白,其中PGEX-NP1为包涵体表达,PGEX-NP2为可溶性和包涵体2种表达形式,因PGEX-NP2蛋白疏水性强、等电点较高,其蛋白大小比理论值偏大;Western blot结果显示2种重组蛋白都具有良好的反应原性。
The study was aimed in cloning and prokaryotic expression of the bovine parainfluenza virus 3 NP gene fragment,at the same time,purification of the obtained protein. According to the NP protein gene sequence of bovine parainfluenza virus 3 （JQ063064） published in GenBank,2 pairs of specific primers were designed and synthetised. Using the RNA extracted from bovine parainfluenza virus 3 NM09 strain virus as the template,the virus was cultured in MDBK cell as the template. Two pieces of NP the gene sequences （NP1,NP2） were amplified by RT-PCR. The NP gene fragments were cloned into prokaryotic expression, vector PGEX-6P=I and transformed into BL21 （DE3）, constructed the GST label recombinant plasmid （PGEX-NP1, PGEX-NP2）. Meanwhile,in order to acquire the soluble protein,we optimized the induced ex- pression conditions. Two kinds of recombinant proteins were identified by SDS-PAGE and Western blot, then they was purified by affinity chromatography. Finally we obtained two kinds of purified proteins with the size of 42.3 ku,38.5 ku,the PGEX-NP1 protein expression form was in inclusion bodies,the PGEX- NP2 protein expression form was in inclusion bodies and soluble protein. Because of the high hydrophobici- ty and isoelectric point of the PGEX-NP2 protein,its size is larger than that of the theoretical value. West- ern blot showed that two kinds of expressed proteins can be recognized by anti-BPIV3 positive serum.
出处
《动物医学进展》
CSCD
北大核心
2014年第2期20-28,共9页
Progress In Veterinary Medicine
基金
国家863计划项目(2011AA10A213)
吉林省特种经济动物疫病防控研究创新团队项目(20121823)
关键词
牛副流感病毒3型
NP基因
原核表达
蛋白纯化
Bovine parainfluenza virus 3
NP gene
prokaryotic expression
protein purification
