[1]顾玲玲,朱善元,成大荣,等.玉型鸭甲型肝炎病毒VP1 基因的原核表达及多克隆抗体的制备[J].江苏农业学报,2017,(01):151-154.[doi:10.3969/j.issn.1000-4440.2017.01.024 ]
　GU Ling-ling,ZHU Shan-yuan,CHENG Da-rong,et al.Prokaryotic expression and antiserum preparation of the VP1 gene of duck hepatitis virus type I[J].,2017,(01):151-154.[doi:10.3969/j.issn.1000-4440.2017.01.024 ]
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玉型鸭甲型肝炎病毒VP1 基因的原核表达及多克隆抗体的制备()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年01期
页码:
151-154
栏目:
畜牧兽医·水产养殖
出版日期:
2017-02-28

文章信息/Info

Title:
Prokaryotic expression and antiserum preparation of the VP1 gene of duck hepatitis virus type I
作者:
顾玲玲12; 朱善元1; 成大荣2; 左伟勇1; 洪伟鸣1; 蔡树东2; 王安平1
（1. 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，江苏 泰州 225300；2. 扬州大学兽医学院，江苏 扬州 225009）
Author(s):
GU Ling-ling12; ZHU Shan-yuan1; CHENG Da-rong2; ZUO Wei-yong1; HONG Wei-ming1; CAI Shu-dong2; WANG An-ping1
(1. Jiangsu Agri-animal Husbandry Vocational College，Veterinary Bio-pharmaceutical，Jiangsu Province Key Laboratory of High-tech Research，Taizhou 225300，China；2. College of Veterinary Medicine，Yangzhou University，Yangzhou 225009，China)
关键词:
Ⅰ型鸭肝炎病毒; VP1基因; 原核表达; 多克隆抗体
Keywords:
duck hepatitis virus type I; VP1 gene; prokaryotic expression; antiserum
分类号:
S852.65+9
DOI:
10.3969/j.issn.1000-4440.2017.01.024
文献标志码:
A
摘要:
根据已经发表的Ⅰ型鸭肝炎病毒VP1基因序列，设计1对特异性引物（上下游分别插入Bam H I和Sac I酶切位点），利用PCR技术扩增出VP1基因，经Bam H I和Sac I双酶切后，将其克隆至原核表达载体pET-32a上，获得重组表达质粒pET-VP1。重组质粒转化感受态细胞E.coli BL21（DE3），重组蛋白经IPTG诱导成功表达。将重组蛋白切胶免疫6周龄的BALB/c小鼠，3次免疫后采血，制备DHV-Ⅰ的多克隆抗体。SDS-PAGE结果显示，成功表达出的重组蛋白分子量约为46 000。Western-blotting分析结果表明，该重组蛋白可与His组氨酸鼠单克隆抗体发生特异性反应，Western-blotting检测抗鼠DHV多抗的结果显示，DHV多抗能与目的蛋白发生特异性反应。以上结果表明，DHV的VP1蛋白在大肠杆菌中成功表达，且制备的DHV多抗能用于VP1蛋白表达的检测。
Abstract:
According to the genome of duck hepatitis virus type I (DHV-I)，one pair of specific primers was designed. The VP1 genes were amplified by PCR, and then were cloned into prokaryotic expression vector pET-32a. The recombinant plasmids were transformed into E.coli BL21（DE3）cells. The proteins were successfully expressed following IPTG induction and detected by the SDS-PAGE. The antiserum against the recombinant protein were produced by immunized BALB/c mouse with recombinant protein. SDS-PAGE showed the approximate molecular weight of the recombinant protein was 46 000. Western-blotting assay revealed that desired proteins could be recognized by the monoclonal antibody against histidine-tagged proteins. Western-blotting results showed that the antiserum could react specifically with desired proteins. These results show that VP1 genes are successfully expressed in E.coli, and the antiserum can be used for detection of VP1 proteins.

参考文献/References:

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[7]唐威华, 张景六, 王宗阳,等. SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因[J]. 植物生理学报, 2000, 26(1):65-69.

备注/Memo

备注/Memo:
收稿日期：2016-04-01 基金项目：国家自然科学基金项目（31302096）；江苏省农业支撑项目（BE2013415）；江苏省六大人才高峰项目（NY-009） 作者简介：顾玲玲（1990-），女，江苏兴化人，硕士研究生，主要从事预防兽医学研究。（E-mail）546374954@qq.com 通讯作者：王安平,（E-mail）wap4017@163.com
更新日期/Last Update: 2017-04-12