鸡传染性法氏囊病病毒VP2蛋白与幽门螺杆菌铁蛋白基因融合PCR扩增及其真核表达载体的构建

doi:10.16431/j.cnki.1671-7236.2019.10.006

中国畜牧兽医 ›› 2019, Vol. 46 ›› Issue (10): 2860-2866.doi: 10.16431/j.cnki.1671-7236.2019.10.006

鸡传染性法氏囊病病毒VP2蛋白与幽门螺杆菌铁蛋白基因融合PCR扩增及其真核表达载体的构建

朱艳平¹, 何勇¹, 刘佳^1,2, 邢瑞林¹, 常乐凯¹, 李润芷^1,3, 岳锋¹, 吴玉苹¹, 李鹏¹, 孙国鹏¹, 张艳芳¹, 王选年^1,2,3

1. 新乡学院生命科学技术学院生物技术研究中心, 新乡 453003;
2. 郑州大学生命科学技术学院, 郑州 450000;
3. 河南科技学院动物科学学院, 新乡 453003

收稿日期:2019-04-29 出版日期:2019-10-20 发布日期:2019-10-21
通讯作者: 王选年 E-mail:wangxuannian@163.com
作者简介:朱艳平(1980-),女,河南宝丰人,博士,副教授,研究方向:分子生物学与分子免疫学,E-mail:yanpingzhu2005@163.com;何勇(1980-),男,河南宁陵人,硕士,讲师,研究方向:分子生物学与分子免疫学,E-mail:yonghe911@163.com
基金资助:
河南省科技攻关项目（182102110221）；新乡学院青年骨干教师资助计划项目（GGJS2016-06）；新乡学院科技创新团队项目（XXUTD20170106）；河南省教育厅自然科学重点研究项目（19A230009）

Fusion PCR of Chicken Infectious Bursal Disease Virus VP2 and Helicobacter pylori Ferritin Fusion Gene and Construction of Its Eukaryotic Expression Vector

ZHU Yanping¹, HE Yong¹, LIU Jia^1,2, XING Ruilin¹, CHANG Lekai¹, LI Runzhi^1,3, YUE Feng¹, WU Yuping¹, LI Peng¹, SUN Guopeng¹, ZHANG Yanfang¹, WAGN Xuannian^1,2,3

1. Center for Biotechnology Research, College of Life Science and Technology, Xinxiang University, Xinxiang 453003, China;
2. School of Life Science, Zhengzhou University, Zhengzhou 450000, China;
3. College of Animal Sciences, Henan Institute of Science and Technology, Xinxiang 453003, China

Received:2019-04-29 Online:2019-10-20 Published:2019-10-21

摘要/Abstract

摘要： 为获得高活性的鸡传染性法氏囊病病毒（infectious bursal disease virus，IBDV）VP2蛋白的重组蛋白，本试验对IBDV VP2蛋白基因与能进行自我组装的幽门螺杆菌（Hp）铁蛋白基因进行融合PCR扩增，获得其融合基因。根据成熟VP2蛋白基因cDNA序列和Hp铁蛋白基因的碱基序列，设计合成2对引物，应用融合PCR技术扩增获得融合基因片段VP2-Fe。将目的基因克隆至pMD18-T载体筛选阳性重组克隆质粒，然后将目的基因亚克隆至真核表达载体pPICZaC后转化大肠杆菌DH5α感受态细胞，筛选获得阳性表达质粒。结果显示，通过两轮PCR扩增出长度为1 824 bp的融合基因VP2-Fe，亚克隆至pMD18-T载体，测序结果显示，融合基因无任何碱基的突变，筛选的阳性质粒命名为pMD-VP2-Fe。双酶切回收目的基因大小为1 824 bp，亚克隆至pPICZaC，PCR和双酶切鉴定出现预期大小的片段，将获得的阳性表达质粒命名为pPICZaC-VP2-Fe。本研究为后期利用真核表达载体获得其融合重组蛋白奠定基础。

关键词: 传染性法氏囊病病毒(IBDV); VP2基因; 幽门螺杆菌; 铁蛋白; 融合PCR

Abstract: In order to obtain the recombinant protein of VP2 protein of infectious bursal disease virus (IBDV) with high activity,the fusion of IBDV VP2 protein and self-assembled Helicobacter pylori (Hp) ferritin gene were amplified by fusion PCR.Two pairs of primers were designed and synthesized according to the DNA sequence of mature VP2 protein gene and the base sequence of Hp ferritin.The fusion gene fragment VP2-Fe was amplified by fusion PCR.The target gene was cloned into pMD18-T vector and the positive recombinant plasmid was screened.Then the target gene was subcloned into eukaryotic expression vector pPICZaC and transformed into competent cells of Escherichia coli DH5α,the positive expression plasmids were screened.The results showed that the fusion gene VP2-Fe with the length of 1 824 bp was amplified by two rounds of PCR and subcloned to pMD18-T vector.Sequencing results showed that there was no mutation in the fusion gene.The positive plasmid was named as pMD-VP2-Fe.The target gene (1 824 bp) was recovered by double digestion and subcloned to pPICZaC.The expected size fragment was identified by PCR and double digestion.The positive expression plasmid was named as pPICZaC-VP2-Fe.This study laid a foundation for the later use of eukaryotic expression vector to obtain its fusion recombinant protein.

Key words: IBDV; VP2 gene; Helicobacter pylori (Hp); ferritin; fusion PCR

中图分类号:

S852.4

朱艳平, 何勇, 刘佳, 邢瑞林, 常乐凯, 李润芷, 岳锋, 吴玉苹, 李鹏, 孙国鹏, 张艳芳, 王选年. 鸡传染性法氏囊病病毒VP2蛋白与幽门螺杆菌铁蛋白基因融合PCR扩增及其真核表达载体的构建[J]. 中国畜牧兽医, 2019, 46(10): 2860-2866.

ZHU Yanping, HE Yong, LIU Jia, XING Ruilin, CHANG Lekai, LI Runzhi, YUE Feng, WU Yuping, LI Peng, SUN Guopeng, ZHANG Yanfang, WAGN Xuannian. Fusion PCR of Chicken Infectious Bursal Disease Virus VP2 and Helicobacter pylori Ferritin Fusion Gene and Construction of Its Eukaryotic Expression Vector[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(10): 2860-2866.

参考文献

[1] XU M Y,LIN S Y,ZHAO Y,et al.Characteristics of very virulent infectious bursal disease viruses isolated from Chinese broiler chickens (2012-2013)[J].Acta Tropica,2015,141(Pt A):128-134.
[2] AMINI K,ZACHAR T,POPOWICH S,et al.Association of increased rate of condemnation of broiler carcasses due to hepatic abnormalities with immunosuppressive diseases in the broiler chicken industry in Saskatchewan[J].Canadian Journal of Veterinary Research,2015,79(4):261-267.
[3] GIMENO I M,SCHAT K A.Virus-induced immunosuppression in chickens[J].Avian Disease,2018,62(3):272-285.
[4] HE X,XIONG Z,YANG L,et al.Molecular epidemiology studies on partial sequences of both genome segments reveal that reassortant infectious bursal disease viruses were dominantly prevalent in Southern China during 2000-2012[J].Archives of Virology,2014,159(12):3279-3292.
[5] LI Y,WANG C,CHENG X,et al.Synonymous codon usage of the VP2 gene of a very virulent infectious bursal disease virus isolate serial passaged in chicken embryos[J].Biosystems,2011,104(1):42-47.
[6] KURUKULSURIYA S,AHMED K A,OJKIC D,et al.Circulating strains of variant infectious bursal disease virus may pose a challenge for antibiotic-free chicken farming in Canada[J].Research in Veterinary Science,2016,108:54-59.
[7] FAN L,WU T,HUSSAIN A,et al.Novel variant strains of infectious bursal disease virus isolated in China[J].Veterinary Microbiology,2019,230:212-220.
[8] JACKWOOD D J,STOUTE S T.Molecular evidence for a geographically restricted population of infectious bursal disease viruses[J].Avian Disease,2013,57(1):57-64.
[9] GEERLIGS H J,ONS E,BOELM G J,et al.Efficacy,safety,and interactions of a live infectious bursal disease virus vaccine for chickens based on strain IBD V877[J].Avian Disease,2015,59(1):114-121.
[10] WANG W,SONG Y,LIU L,et al.Neutralizing-antibody-mediated protection of chickens against infectious bursal disease via one-time vaccination with inactivated recombinant Lactococcus lactis expressing a fusion protein constructed from the RCK protein of Salmonella enterica and VP2 of infectious bursal disease virus[J].Microbial Cell Factories,2019,18(1):21.
[11] HERNÁNDEZ M,TOMÁS G,MARANDINO A,et al.Genetic characterization of South American infectious bursal disease virus reveals the existence of a distinct worldwide-spread genetic lineage[J].Avian Pathology,2015,44(3):212-221.
[12] DE FRAGA A P,GRÄF T,COLTRO V P,et al.Phylodynamic analyses of Brazilian antigenic variants of infectious bursal disease virus[J].Infection Genetics and Evolution,2019,73:159-166.
[13] MOHAMED M A,ELZANATY K E,BAKHIT B M,et al.Genetic characterization of infectious bursal disease viruses associated with gumboro outbreaks in commercial broilers from Asyut province,Egypt[J].ISRN Veterinary Science,2014,9(1):91641-916421.
[14] 高祥,李辉,张礼洲,等.传染性法氏囊病病毒超强毒株衣壳蛋白的可溶性表达、纯化与活性分析[J].中国畜牧兽医,2017,44(7):2096-2102. GAO X,LI H,ZHANG L Z,et al.Soluble expression,purification and activity analysis of capsid protein of very virulent infectious bursal disease virus[J].China Animal Husbandry & Veterinary Medicine,2017,44(7):2096-2102.(in Chinese)
[15] 王洁琼,赵玉杰,周云飞,等.传染性法氏囊病病毒C4毒株VP2基因的原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(12):3585-3591. WANG J Q,ZHAO Y J,ZHOU Y F,et al.Prokaryotic expression and bioinformatics analysis of VP2 gene of infectious bursal disease virus C4 strain[J].China Animal Husbandry & Veterinary Medicine,2017,44(12):3585-3591.(in Chinese)
[16] 杨凤,黎先伟,孙敏华,等.传染性法氏囊病病毒VP2基因在昆虫细胞中的表达[J].中国畜牧兽医,2013,40(9):65-69. YANG F,LI X W,SUN M H,et al.Expression of VP2 gene of infectious bursal disease virus in insect cells[J].China Animal Husbandry & Veterinary Medicine,2013,40(9):65-69.(in Chinese)
[17] 郭振环,马霞,王恬,等.蜂胶黄酮对宿主细胞抗病毒能力的影响[J].中国畜牧兽医,2016,43(8):2190-2195. GUO Z H,MA X,WANG T,et al.Effects of propolis flavone on antiviral ability of host cell[J].China Animal Husbandry & Veterinary Medicine,2016,43(8):2190-2195.(in Chinese)
[18] EBRAHIMI M M,SHAHSAVANDI S,SHAYAN P.TIR-TLR7 as a molecular adjuvant:Simultaneous enhancing humoral and cell-mediated immune responses against inactivated infectious bursal disease virus[J].Viral Immunology,2019,32(6):252-257.
[19] LI C Q,SOISTMAN E,CARTER D C.Ferritin nanoparticle technology.A new platform for antigen presentation and vaccine development[J].Industrial Biotechnology,2006,2(2):143-147.
[20] KANEKIYO M,WEI C,YASSINE H M,et al.Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies[J].Nature,2013,499(7456):102-106.
[21] HAN J A,KANG Y J,SHIN C,et al.Ferritin protein cage nanoparticles as versatile antigen delivery nanoplatforms for dendritic cell (DC)-based vaccine development[J].Nanomedicine,2014,10(3):561-569.
[22] DA COSTA B,CHEVALIER C,HENRY C,et al.The capsid small peptides arising from maturation process of pVP2[J].Journal of Virology,2002,76(5):2393-2402.
[23] 韩玉婷.鸡传染性法氏囊病病毒NB株VP2成熟蛋白基因在毕赤酵母中的表达[D].新乡:河南科技学院,2016. HAN Y T.Expression of VP2 gene of IBDV NB strain in Pichia pastoris[D].Xinxiang:Henan Institute of Science and Technology,2016.(in Chinese)
[24] 邢瑞林.SUMO系统表达IBDV NB株VP2 S-P可溶性蛋白[D].郑州:郑州大学,2018. XING R L.VP2 S-P soluble protein of IBDV NB strain expressed by SUMO system[D].Zhengzhou:Zhengzhou University,2018.(in Chinese)

鸡传染性法氏囊病病毒VP2蛋白与幽门螺杆菌铁蛋白基因融合PCR扩增及其真核表达载体的构建

Fusion PCR of Chicken Infectious Bursal Disease Virus VP2 and Helicobacter pylori Ferritin Fusion Gene and Construction of Its Eukaryotic Expression Vector

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]	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.
[2]	刘家兴, 韩雪英, 张鑫茹, 邓嘉昕, 姚伦广, 刘阳坤. 携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析[J]. 中国畜牧兽医, 2023, 50(4): 1567-1574.
[3]	何献铭, 任广彩, 叶俊贤, 兰虹, 刘郁夫, 熊挺, 杨泽坤, 徐婷, 陈瑞爱. 1株传染性法氏囊病病毒的分离鉴定及VP2基因序列分析[J]. 中国畜牧兽医, 2022, 49(3): 1005-1014.
[4]	谭斌, 王超, 王岩, 张淑琴. 果子狸源细小病毒分离鉴定及VP2基因遗传进化分析[J]. 中国畜牧兽医, 2022, 49(10): 4012-4018.
[5]	马英, 呼尔查, 李建龙, 郝蕴伟, 李敏, 范士龙, 韦丽婷, 陈宋琴, 张婷, 郭庆勇. 铁蛋白1基因在边缘革蜱各发育龄期、吸血期及不同器官中的表达分析[J]. 中国畜牧兽医, 2021, 48(5): 1841-1848.
[6]	张珺妍, 谭宏超, 陈佩佩, 王晓娜, 姜艳平, 崔文, 乔薪瑗, 王丽, 周晗, 徐义刚, 李一经, 唐丽杰. 表达牛乳铁蛋白肽的重组乳酸乳球菌微胶囊制备工艺的优化[J]. 中国畜牧兽医, 2021, 48(12): 4607-4617.
[7]	孟维金, 范阔海, 常熊熊, 尹伟, 李宏全, 王志瑞. 鸡传染性法氏囊病病毒VP2与白喉毒素截短体DT390融合蛋白的真核表达及免疫原性检测[J]. 中国畜牧兽医, 2021, 48(12): 4652-4660.
[8]	王彦伟, 张素玲, 吴芃, 耿笑林, 黄玉欣, 李鹏昊, 王孟月, 李向东, 逄文强, 田克恭. 鸡传染性法氏囊病病毒rVP2蛋白高效组装亚病毒颗粒的鉴定[J]. 中国畜牧兽医, 2020, 47(6): 1677-1684.
[9]	解伟纯, 王玉峰, 皮若冰, 赵东方, 姜艳平, 崔文, 乔薪瑗, 王丽, 周晗, 徐义刚, 李一经, 唐丽杰. 表达猪乳铁蛋白肽重组屎肠球菌对断奶仔猪生长性能的影响及其抗ETEC感染效果研究[J]. 《中国畜牧兽医》, 2018, 45(12): 3408-3418.
[10]	王建珏, 史怀平, 马功珍, 黄江涛, 吕明, 张志飞. 奶山羊LF基因CDS区克隆、生物信息学分析及其乳中含量测定[J]. 《中国畜牧兽医》, 2018, 45(10): 2805-2813.
[11]	王洁琼, 赵玉杰, 周云飞, 黄宗梅, 陈盼盼, 周薇帆, 刘琳, 李新生. 传染性法氏囊病病毒C4毒株VP2基因的原核表达及生物信息学分析[J]. 《中国畜牧兽医》, 2017, 44(12): 3585-3591.
[12]	林筱璐, 林秀娇, 王秀爱, 刘凤军, 庄益芬, 张兴锋, 李鸿翔, 洪志勇, 刘芳, 张文昌. 不同因素对猪卵母细胞体外成熟培养的影响[J]. , 2014, 41(10): 205-209.
[13]	李建波, 江明锋, 王永. 藏绵羊乳腺乳铁蛋白的克隆及序列分析[J]. , 2013, 40(9): 86-90.
[14]	张娟霞, 孙鹏, 杨永新, 卜登攀, 周凌云, 雒秋江. 牛奶中抗氧化物质研究进展[J]. , 2012, 39(7): 142-146.
[15]	付强;杨丽;刘振方;张明;卢克焕. 水牛卵泡液液相色谱分离及差异蛋白质分析[J]. , 2012, 39(4): 64-67.