犬细小病毒NY株VP2蛋白多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2016.07.001

《中国畜牧兽医》---唯一指定的官方网站 ›› 2016, Vol. 43 ›› Issue (7): 1659-1666.doi: 10.16431/j.cnki.1671-7236.2016.07.001

• 生物技术 • 下一篇

犬细小病毒NY株VP2蛋白多克隆抗体的制备与鉴定

毛倩倩¹, 周灵^2,3, 唐青海¹, 卜宾¹, 唐存多¹, 焦铸锦¹, 姚伦广¹, 阚云超¹, 杨建伟¹, 崔尚金³

1. 南阳师范学院, 南阳市兽医生物工程技术研究中心, 河南省伏牛山昆虫生物学重点实验室, 昆虫生物反应器河南省工程实验室, 南阳 473061;
2. 吉林农业大学动物科学技术学院, 长春 130118;
3. 中国农业科学院北京畜牧兽医研究所, 北京 100193

收稿日期:2016-01-11 出版日期:2016-07-20 发布日期:2016-07-22
通讯作者: 唐青海, 崔尚金 E-mail:qinghaitang109@126.com;cuishangjin@caas.cn
作者简介:毛倩倩(1988-),女,河南安阳人,硕士生,研究方向:生物制药与疫苗工程,E-mail:601681088@qq.com
基金资助:
河南省重点科技攻关项目（142102110101）；河南省高等学校重点科研项目（16A230023）；南阳师范学院引进人才专项项目（70640）

Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody

MAO Qian-qian¹, ZHOU Ling^2,3, TANG Qing-hai¹, BU Bin¹, TANG Cun-duo¹, JIAO Zhu-jin¹, YAO Lun-guang¹, KAN Yun-chao¹, YANG Jian-wei¹, CUI Shang-jin³

1. Center for Nanyang Veterinary Biological Engineering Technology, Henan Provincial Engineering Laboratory of Insect Bio-reactor, Henan Provincial Key Laboratory of Insect Biology in Funiu Moutain, Nanyang Normal University, Nanyang 473061, China;
2. Animal Science and Technology College, Jilin Agricultural University, Changchun 130118, China;
3. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2016-01-11 Online:2016-07-20 Published:2016-07-22

摘要/Abstract

摘要：

试验旨在制备犬细小病毒（canine parvovirus，CPV）VP2蛋白多克隆抗体。构建pET28a-CPV-VP2重组表达质粒转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导，SDS-PAGE鉴定融合蛋白的表达，纯化目的蛋白与佐剂混合、乳化制备后作为免疫原，免疫家兔制备VP2蛋白多克隆抗体；采用免疫过氧化物酶单层细胞染色法（immunoperoxidase monolayer assay，IPMA）检测抗体的免疫活性、抗体滴度、病毒滴度及中和活性。结果表明，重组VP2蛋白（rVP2）以包涵体形式存在，分子质量约为72 ku；所制备的多克隆抗体滴度为1 600倍，病毒滴度为10⁷ TCID₅₀ /mL，中和效价为1∶2 884，该抗体与体外培养的CPV呈特异性反应，CPV VP2蛋白的多克隆抗体免疫活性和特异性良好，且中和活性高，为CPV基因工程疫苗的研究和临床治疗奠定了基础。

关键词: 犬细小病毒; VP2基因; 多克隆抗体; 免疫活性

Abstract:

This study was aimed to prepare canine parvovirus (CPV) VP2 protein polyclonal antibody.The recombinant expression vector pET28a-CPV-VP2 was constructed and transfromed into E.coli BL21 (DE3),the expression of recombinant proteins was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The target protein was purified and emulsify with adjuvant,the prepared immunogen was inoculated into rabbit by subcutaneous injections to prepare of VP2 protein specific polyclonal antibody.The immuno-activity,titers,neutralization titers of the prepared polyclonal antibody were determined by immunoperoxidase monolayer assay (IPMA).The results showed that the expressed recombinant protein VP2 (rVP2) existed in the form of inclusion body with a molecular weight of 72 ku.The prepared polyclonal antibody titer was 1 600 dilution,the virus titer was 10⁷ TCID₅₀/mL,the neutralizing titer was 1∶2 884.The antibodies showed specific reaction with CPV.In conclusion,rVP2 specific polyclonal antibody showed wonderful immunocompetence,specificity and neutralizing activity,providing foundation for the development of genetic vaccine and clinical therapeutic method.

Key words: caine parvirus; VP2 gene; polyclonal antibody; immunocompetence

中图分类号:

S852.65⁺5

毛倩倩, 周灵, 唐青海, 卜宾, 唐存多, 焦铸锦, 姚伦广, 阚云超, 杨建伟, 崔尚金. 犬细小病毒NY株VP2蛋白多克隆抗体的制备与鉴定[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2016, 43(7): 1659-1666.

MAO Qian-qian, ZHOU Ling, TANG Qing-hai, BU Bin, TANG Cun-duo, JIAO Zhu-jin, YAO Lun-guang, KAN Yun-chao, YANG Jian-wei, CUI Shang-jin. Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody[J]. , 2016, 43(7): 1659-1666.

参考文献

[1] 刘静,赵学刚,陆江,等.犬细小病毒流行病学调查[J].中国畜牧兽医,2013,40(3):199-201.
[2] Welborn L V,DeVries J G, Ford R, et al.2011 AAHA canine vaccination guidelines[J].J Am Anim Hosp Assoc,2011,47(5):1-42.
[3] Apple M J, Scott F W, Carmichael L E.Isolation and immunisation studies of a caine parco-like virus from dogs with haemorrhagic enteritis[J].Vet Rec,1979,105(8):156-159.
[4] Truyen U, Evermann J F, Vieler E, et al.Evolution of canine parvovirus involved loss and gain of feline host range[J].Virology,1996,215(2):186-189.
[5] Parrish C R, Aquadro C F, Strassheim M L, et al.Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus[J].J Virol,1991,65(12):6544-6552.
[6] Buonavoglia C, Martella V, Pratelli A, et al.Evidence for evolution of canine parvovirus type 2 in Italy[J].J Gen Virol,2001,82(12):3021-3025.
[7] Decaro N, Desario C, Billi M, et al.Western European epidemiological survey for parvovirus and coronavirus infections in dogs[J].Vet J,2011,187(2):195-199.
[8] Touihri L, Bouzid I,Daoud R, et al.Molecular characterization of canine parvovirus-2 variants circulating in Tunisia[J].Virus Genes,2009,38(2):249-258.
[9] Nakamura M, Tohya Y,Miyazawa T, et al.A novel antigenic variant of canine parvovirus from a Vietnamese dog[J].Arch Virol,2004,149(11):2261-2269.
[10] Hong C, Decaro N, Desario C,et al.Occurrence of canine parvovirus type 2c in the United States[J].J Vet Diagn Invest,2007,19(5):535-539.
[11] Perez R, Francia L, Romero V.First detection of canine parvovirus type 2c in South America[J].Veterinary Microbiology,2007,124:147-152.
[12] Geng Y F,Guo D H, Li C Q, et al.Co-circulation of the rare CPV-2c with unique Gln370Ala substitution,abd new CPV-2a with high prevalence and variation in Heilongjiang province,Northeast China[J].PLoS One,2016,10(9):e0137288.
[13] Parker J S, Murphy W J, Wang D.Canine and feline parvoviruses can use human or feline transferrin receptors to bind enter and infect cells[J].J Virol,2001,75(8):3896-3902.
[14] Hueffer K, Palermo L M, Parrish C R.Parvovirus infection of cells by using variants of the feline transferrin receptor altering clathrin-mediated endocytosis,membrane domain localization,and capsid-binding domains[J].J Virol,2004,78(11):5601-5611.
[15] Hueffer K,Govindasamy L, Agbandje M.Combinations of two capsid regions controlling canine host range determine canine transferrin receptor binding by canine and feline parvoviruses[J].Virology,2003,77(18):10099-10105.
[16] Jeoung S Y,Ahn S J,Kim H.Genetic analysis of VP2 gene of canine parvovirus isolates in Korea[J].J Vet Med Science,2008,70:719-722.
[17] 刘杉杉,赵亚荣,张超范,等.猪细小病毒抗体IPMA检测方法的建立及应用[J].中国兽医科学,2011,41(4):387-391.
[18] Direksin K, Joo H,Goyal S M.An immunoperoxidase monolayer assay for the detection of antibodies against swine influenza virus[J].J Vet Diagn Invest,2002,14(2):169-171.
[19] Streck A F, Ruster D, Truyen U.An updated TaqMan Real-time PCR for canine and feline parvoviruses[J].Journal of Virological Methods,2013,193(1):6-8.
[20] 张立媛.犬细小病毒YBYJ株的分离鉴定及VP2基因克隆与多克隆抗体的制备[D].延边:延边大学,2015.

犬细小病毒NY株VP2蛋白多克隆抗体的制备与鉴定

Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[2]	欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2487-2495.
[3]	王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.
[4]	胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.
[5]	王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.
[6]	牛小杰, 徐明国, 肖莉, 刘文豪, 陈创夫, 王勇. 鸡白痢沙门氏菌IPAJ蛋白的表达纯化、多克隆抗体制备及ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(3): 1218-1228.
[7]	张梦杨, 何健, 刘阳坤, 姚伦广. 非洲猪瘟病毒p37蛋白生物信息学分析及其多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(1): 300-309.
[8]	朱丽霖, 王后坤, 陈雪阳, 刘晶, 梁雄燕, 方春, 杨玉莹. 鸡IFIT5对J亚群禽白血病病毒在DF-1细胞内复制的影响[J]. 中国畜牧兽医, 2023, 50(1): 310-316.
[9]	任泽恒, 皮雪磊, 夏安然, 孙悦, 胡昌辉, 任桂萍. 抗犬细小病毒纳米抗体的制备及其中和活性鉴定[J]. 中国畜牧兽医, 2022, 49(9): 3539-3548.
[10]	肖洋洋, 马忠臣, 李芮芮, 陈创夫, 郑炜, 王勇, 王鹏雁. 布鲁氏菌分泌蛋白BspI生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(8): 3112-3121.
[11]	王炜, 韩慧慧, 丁乃峥, 何成强. 牛病毒性腹泻病毒E0蛋白的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(8): 3131-3139.
[12]	刘铭, 张永宁. 猪丁型冠状病毒N蛋白原核表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2022, 49(7): 2698-2707.
[13]	许春梅, 邵明珠, 王心悦, 林佳迪, 郭建雄, 张祥胤, 童德文, 梁瑞英, 赵晓民. 非洲猪瘟病毒D250R蛋白生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2022, 49(12): 4745-4755.
[14]	刘超, 王后坤, 朱丽霖, 刘晶, 梁雄燕, 方春, 杨玉莹. 鸡RNF165蛋白多克隆抗体制备及其生物信息学分析[J]. 中国畜牧兽医, 2022, 49(11): 4159-4167.
[15]	张天爱, 李婷婷, 陶晓莉, 李藤菲, 林家锋, 胡思漫, 刘文秀, 佟伟, 李永刚. 猪流行性腹泻病毒S蛋白原核表达及多克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2022, 49(11): 4383-4391.