[1]郭芸芸,张碧成,孙小涵,等.Fh8明显增强PCV2 Cap蛋白的可溶性表达及抗原性[J].江苏农业学报,2018,(02):356-360.[doi:doi:10.3969/j.issn.1000-4440.2018.02.019]
　GUO Yun-yun,ZHANG Bi-cheng,SUN Xiao-han,et al.Fasciola hepatica 8 significantly enhances soluble expression of Cap from PCV2 and its immunogenicity[J].,2018,(02):356-360.[doi:doi:10.3969/j.issn.1000-4440.2018.02.019]
点击复制

Fh8明显增强PCV2 Cap蛋白的可溶性表达及抗原性()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2018年02期
页码:
356-360
栏目:
畜牧兽医·水产养殖
出版日期:
2018-04-25

文章信息/Info

Title:
Fasciola hepatica 8 significantly enhances soluble expression of Cap from PCV2 and its immunogenicity
作者:
郭芸芸1; 2; 张碧成1; 孙小涵1; 汪伟1; 何孔旺1; 牛家强2; 张雪寒1
（1.江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/江苏省食品质量安全重点实验室——省部共建国家重点实验室培育基地，江苏南京210014；2.西藏农牧学院动物科学学院，西藏林芝860000）
Author(s):
GUO Yun-yun1; 2; ZHANG Bi-cheng1; SUN Xiao-han1; WANG Wei1; HE Kong-wang1; NIU Jia-qiang2; ZHANG Xue-han1
（1.Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry/Jiangsu Key Laboratory for Food Quality and Safety——State Key Laboratory Cultivation Base of Ministry of Science and Technology, Nanjing 210014，China;2.Academy of Animal Sciences, Xizang Agriculture and Animal Husbandry College, Linzhi 860000，China）
关键词:
猪圆环病毒2型; 结构蛋白; Fh8; 可溶性表达; 抗原性
Keywords:
porcine circovirus type2(PCV2); structural protein; Fh8; soluble expression; antigenicity
分类号:
S852.65+9.2
DOI:
doi:10.3969/j.issn.1000-4440.2018.02.019
文献标志码:
A
摘要:
猪圆环病毒2型（Porcine circovirus type 2, PCV2）的主要功能蛋白Cap蛋白，很难在体外获得可溶性表达。本研究旨在以Fh8蛋白为融合标签实现PCV2 ORF2基因在大肠杆菌中的可溶性表达，以小鼠评价可溶性Cap蛋白的抗原性。以病毒基因组为模板，扩增ORF2靶基因，插入pCold原核表达质粒，成功构建的pCold-ORF2阳性质粒转入大肠杆菌BL21中。利用SDS-PAGE分析Cap蛋白的可溶性表达情况，利用Western blotting鉴定Cap蛋白的反应原性。重组蛋白与佐剂乳化制备免疫原，免疫Balb/c小鼠，ELISA方法检测血清中Cap抗体，利用PCR检测组织脏器中病毒载量，以评估可溶性Cap蛋白的免疫原性。PCR结果表明，Fh8 和PCV2 ORF2基因被成功扩增。SDS-PAGE结果表明Fh8标签显著提高了ORF2在大肠杆菌中的可溶性表达，分子量 35 000。Western blotting结果显示，猪PCV2阳性血清能够特异性的识别可溶性Cap蛋白。小鼠一次接种Cap蛋白28 d后，抗体全部转阳，免于PCV2的攻击。表明通过新型小标签实现了PCV2 ORF2基因的可溶性表达，且具有良好的免疫原性。
Abstract:
Cap protein is the main functional protein of porcine circovirus type 2 (PCV2), but it is difficult to obtain soluble expression in vitro. Our object was to realize soluble expression of PCV2 ORF2 gene by Fh8-tagged in Escherichia coli , and to evaluate the antigenicity of the recombinant Cap protein in mice. PCV2 DNA was used as template to amplify ORF2 gene and was cloned into prokaryotic expression vector pCold. The resultant pCold-ORF2 plasmid was transformed into E.coli BL21. SDS-PAGE and Western blotting assay were used to analyze the solubility of the recombinant protein and to identify its reactivity, respectively. The recombinant protein and adjuvant were emulsified to prepare vaccine to immune Balb/c mice. The specific antibody of Cap recombinant protein in sera was detected by ELISA method, and virus loading in tissues was detected by PCR. So, the immunogenicity of Cap protein could be assessed. The PCR results showed that Fh8 and PCV2 ORF2 genes were successfully amplified. SDS-PAGE results showed that soluble expression of ORF2 in E. coli was significantly increased by Fh8 tag, and the molecular weight was 35 000. Western blotting results showed that the soluble Cap protein was recognized specifically by porcine PCV2 positive sera. Mice inoculated with the Cap protein vaccine produced high antibody titers at 28 d after initial immunization and could be well protected from PCV2 attack. The soluble expression of PCV2 ORF2 gene was achieved by a novel small label, and the Cap vaccine had good immunogenicity.

参考文献/References:

[1]曹东阳，王小敏，钱爱东，等． 江苏省及周边地区猪圆环病毒Ⅱ型（PCV2） 分子流行病学调查［J］ ．江苏农业学报，2016,32(2):390-398.
[2]SORDEN S D. Update on porcine circovirus and postweaning multisystemic wasting syndrome (PMWS) [J].Swine Health Prod, 2000, 8(3):133-136.
[3]杨剑，尹 丹，郝祝兵，等. 猪圆环病毒2型体外诱导RAW264.7细胞氧化应激模型的建立[J]. 南方农业学报，2017，48(1)：151-157.
[4]郭广富,曹军平,朱爱萍,等. 猪圆环病毒 2 型泰州株的分离鉴定[J]. 江苏农业科学,2016,44(4):294-295.
[5]代振江，王伟丞，曾智勇，等. 猪圆环病毒基因组结构与功能研究进展[J]. 畜牧与兽医，2016（3）：151-154.
[6]NAWAGITGUL P, MOROZOV I, BOLIN S R, et al. Open reading frame 2 of porcine circovirus type 2 encodesa major capsid protein [J].Journal of Gene Virology, 2000, 81(9):2281-2287.
[7]张挺杰，刘星，孙涛，等. 表达猪圆环病毒2型ORF2基因的重组猪繁殖与呼吸综合征病毒的构建与鉴定[J]. 病毒学报，2015，31（1）：65-73.
[8]赵晓云，乔绪稳，陈瑾，等. 利用E.coli表达猪圆环病毒2型Cap蛋白生产病毒样颗粒疫苗[J].中国农业科学，2015，48（5）：976-986.
[9]吴洋，李庆伟，杨娜娜. 猪PCV2 ORF2蛋白ELISA检测方法的建立及猪场血清抗体检测分析[J]. 免疫学杂志，2015（1）:71-73.
[10]COSTA S J, COELHO E, FRANCO L, et al. The Fh8 tag: a fusion partner for simple and cost-effective protein purification in Escherichia coli [J]. Protein Expression and Purification, 2013, 92：163-170.
[11]COSTA S J, ALMEIDA A, CASTRO A, et al. The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology [J].Applied Microbiology and Biotechnology, 2013, 97(15):6779-6791.
[12]COSTA S J, ALMEIDA A, CASTRO A, et al. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system [J]. Frontier in Microbiology, 2014,5(63):1-20.
[13]马涛，王先炜，姜平. 不同佐剂对猪圆环病毒2型灭活抗原免疫效果的影响[J].中国兽医科学，2011，41（6）：593-600.
[14]赵路易，杨增岐，刘建营. 猪圆环病毒2型感染裸鼠及BALB/c小鼠的研究[J].中国兽医科学，2007，37（7）：568-573.
[15]GUO X Q, WANG L Q, QIAO H, et al. Enhancement of the immunogenicity of a porcine circovirus type 2 DNA vaccine by a recombinant plasmid co-expressing capsid protein and porcine interleukin-6 in mice [J]. Microbiology Immunology, 2015，59(3):174-180.

相似文献/References:

[1]徐悦,常晨,侯继波,等.PCV2 病毒生物矿化条件的优化[J].江苏农业学报,2017,(01):159.[doi:10.3969/j.issn.1000-4440.2017.01.026 ]
　XU Yue,CHANG Chen,HOU Ji-bo,et al.Optimization of biomineralization conditions of porcine circovirus type 2(PCV2) viral particles[J].,2017,(02):159.[doi:10.3969/j.issn.1000-4440.2017.01.026 ]

备注/Memo

备注/Memo:
收稿日期：2017-09-05 基金项目：江苏省科技自主创新基金项目[CX(15)1060]；国家自然科学基金项目（31572503） 作者简介：郭芸芸（1994-），女，山西长治人，硕士研究生，从事动物重大疫病的防控技术研究。（E-mail）1692373770@qq.com 通讯作者：张雪寒，（E-mail）： liuxuehan1996@hotmail.com
更新日期/Last Update: 2018-05-04