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1型鸭甲型肝炎病毒3D基因的原核表达与多抗制备
王安平,朱善元,吴双
0
(江苏农牧科技职业学院,江苏省兽用生物制药高技术研究重点实验室,泰州 225300)
摘要:
根据血清1型鸭甲型肝炎病毒(DHAV-1) SH株3D基因序列设计并合成1对特异性引物,通过RT-PCR方法扩增3D基因,将其克隆入原核表达载体pET-32a,转化大肠杆菌BL21(DE3),在IPTG的诱导培养下重组蛋白获得了成功表达。SDS-PAGE显示重组蛋白的分子量约为68 kD,Western blot分析显示该重组蛋白能与多聚组氨酸标签单抗发生特异性反应。将切胶后纯化的目的蛋白免疫ICR小鼠,制备针对重组蛋白的多抗血清,Western blot结果显示制备的抗血清能够与DHAV-1感染的SPF鸡胚尿囊液总蛋白发生特异反应。以上结果表明,3D基因在大肠杆菌中获得了成功表达,且制备的多抗血清可以用于3D蛋白的检测,为3D蛋白的功能研究奠定了基础。
关键词:  鸭甲型肝炎病毒  3D基因  原核表达  抗血清
DOI:10.13610/j.cnki.1672-352x.20150626.006
投稿时间:2014-07-31
基金项目:国家自然科学基金(31302096), 江苏省农业支撑项目(BE2013415), 江苏省自然科学基金项目(BK2011536), 江苏省”企业博士集聚计划”和江苏省”六大人才高峰项目”(NY-009)共同资助。
Prokaryotic expression and antiserum preparation of the 3D gene of Duck hepatitis A virus type-1
WANG Anping,ZHU Shanyuan,WU Shuang
(Veterinary Bio-pharmaceutical, Jiangsu Province Key Laboratory of High-tech Research, Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300)
Abstract:
According to the genome sequences of duck hepatitis A virus type-1(DHAV-1), one pair of specific primers was designed to amplify the 3D gene using RT-PCR. The amplified fragment was cloned into prokaryotic expression vector pET-32a and the recombinant plasmid was transformed into BL21(DE3) E.coli. Recombinant proteins were successfully expressed following IPTG induction. SDS-PAGE showed that the recombinant expression protein was about 68 kD. Western blot assay revealed that the desired proteins could be recognized by the monoclonal antibody against histidine-tagged proteins. The antiserum against the recombinant protein was produced by the immunized ICR mouse with recombinant proteins. Western blot results showed that the antiserum could react specifically with DHAV-1 allantoic fluid. These results showed that the 3D gene was successfully expressed in E.coli and the antiserum could be used for detection of the 3D protein, which would lay a foundation for function study of the 3D protein.
Key words:  duck hepatitis A virus type  3D gene  prokaryotic expression  antiserum

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