表达水泡性口炎病毒双血清型G蛋白重组腺病毒的构建及对小鼠的免疫原性分析

doi:10.11843/j.issn.0366-6964.2015.12.014

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (12): 2227-2234.doi: 10.11843/j.issn.0366-6964.2015.12.014

表达水泡性口炎病毒双血清型G蛋白重组腺病毒的构建及对小鼠的免疫原性分析

薛晓娟，金红岩，赵玲娜，隋修锟，Zheney Makay，李刚^*

（中国农业科学院北京畜牧兽医研究所动物营养学国家重点实验室/农业部兽用药物与兽医生物技术北京科学观测实验站，北京100193）

收稿日期:2015-05-07 出版日期:2015-12-23 发布日期:2015-12-18
通讯作者: 李刚，教授，E-mail：ligang03@caas.cn，Tel：010-62818775
作者简介:薛晓娟（1989-），女，山东聊城人，硕士生，主要从事动物重大疫病的诊断与防治研究，E-mail：xiaojuanxue@163.com
基金资助:
国家科技支撑计划项目（2013BAD12B05）；国家自然科学基金项目（31172342；31472203）；转基因重大专项（2014ZX0801203B）；国际合作项目（D32025/17453）；国家公益行业项目（200903037-2）；创新团队基金（ASTIP-IAS15）

Construction and Immunogenicity of Recombinant Adenovirus Expressing Two Serotypes of Vesicular Stomatitis Virus G Proteins in Mice

XUE Xiao-juan，JIN Hong-yan，ZHAO Ling-na，SUI Xiu-kun，Zheney Makay，LI Gang^*

(State Key Laboratory of Animal Nutrition/Beijing Scientific Observation and Experiment Station for Veterinary Drug and Veterinary Biotechnology of Ministry of Agriculture，Institute of Animal Science，Chinese Academy of Agricultural Sciences，Beijing 100193，China)

Received:2015-05-07 Online:2015-12-23 Published:2015-12-18

摘要/Abstract

摘要：

旨在以人5型复制缺陷型腺病毒为表达载体，构建表达水泡性口炎病毒(VSV)双血清型G蛋白的重组腺病毒。利用融合PCR技术扩增VSV-IN-G-NJ-G基因并将其克隆至腺病毒穿梭载体pacAd-CMV K-NpA中。通过Pac Ⅰ酶线性化后，与同样经PacⅠ酶线性化的骨架载体pacAd5 9.2-100共转染AAV-293细胞，获得重组腺病毒rAd-VSV-IN-G-NJ-G。该重组腺病毒于AAV-293细胞连续传代至20代效价稳定。经间接免疫荧光和Western blot检测，G蛋白获得正确表达。将重组腺病毒接种小鼠，利用间接ELISA及病毒中和试验检测其体液免疫水平，结果显示可诱导产生VSV特异性抗体，其中和抗体水平在1∶32；利用淋巴细胞增殖试验检测细胞免疫水平，结果表明可以引起接种小鼠强烈的淋巴细胞增殖。构建的重组腺病毒免疫小鼠后可以引起一定的体液免疫和细胞免疫反应，为表达VSV糖蛋白重组腺病毒疫苗的深入研究奠定了基础。

关键词: VSV G蛋白, 重组腺病毒, 免疫反应, 小鼠

Abstract:

The purpose of this study was to construct the recombinant adenovirus expressing two serotypes of vesicular stomatitis virus(VSV) G proteins with human adenovirus expression vector.The gene VSV-IN-G-NJ-G was amplified by fusion-PCR and cloned into the adenovirus shuttle vector pacAd-CMV K-NpA.The recombinant shuttle plasmid and adenovirus backbone plasmid linearized by digestion with PacⅠ，and then were transfacted into AAV-293 cells to obtain recombinant adenovirus rAd-VSV-IN-G-NJ-G.The recombinant adenovirus was stable in AAV-293 cells after serial passage to 20 generations.By indirect immunofluorescence and western blot detection，G proteins were expressed in recombinant adenovirus.The mice were inoculated with recombinant adenovirus for evaluating the humoral immune responses by indirect ELISA and neutralization experiment.The results showed that the recombinant adenovirus could induce VSV specific antibodies and the neutralizing antibody level of inoculated group was 1：32.The result of cellular immune showed that the recombinant adenovirus can cause a strong lymphocyte proliferation of the inoculated mice.These results suggested that the recombinant adenovirus can cause a certain degree of humoral and cellular immune responses in inoculated mice.The research laid foundations for further studies of VSV gene-engineering vaccines.

Key words: VSV G protein, recombinant adenovirus, immunogenicity, mice

中图分类号:

S852.659.5

薛晓娟，金红岩，赵玲娜，隋修锟，Zheney Makay，李刚. 表达水泡性口炎病毒双血清型G蛋白重组腺病毒的构建及对小鼠的免疫原性分析[J]. 畜牧兽医学报, 2015, 46(12): 2227-2234.

XUE Xiao-juan，JIN Hong-yan，ZHAO Ling-na，SUI Xiu-kun，Zheney Makay，LI Gang. Construction and Immunogenicity of Recombinant Adenovirus Expressing Two Serotypes of Vesicular Stomatitis Virus G Proteins in Mice[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2227-2234.

参考文献

［1］HEO E J，LEE H S，JEOUNG H Y，et al.Development of a blocking ELISA using a recombinant glycoprotein for the detection of antibodies to vesicular stomatitis New Jersey virus［J］.J Virol Methods，2010，164(1-2)：96-100.
［2］MIRE C E，WHITT M A.The protease-sensitive loop of the vesicular stomatitis virus matrix protein is involved in virus assembly and protein translation［J］. Virology，2011，416(1-2)：16-25.
［3］FERRIS N P，CLAVIJO A，YANG M，et al.Development and laboratory evaluation of two lateral flow devices for the detection of vesicular stomatitis virus in clinical samples［J］.J Virol Methods，2012，180(1-2)：96-100.
［4］HOUSE J A，HOUSE C，DUBOURGET P，et al.Protective immunity in cattle vaccinated with a commercial scale，inactivated，bivalent vesicular stomatitis vaccine［J］.Vaccine，2003，21(17-18)：1932-1937.
［5］VELAZQUEZ-SALINAS L，PAUSZEK S J，ZARATE S，et al.Phylogeographic characteristics of vesicular stomatitis New Jersey viruses circulating in Mexico from 2005 to 2011 and their relationship to epidemics in the United States［J］. Virology，2014，449：17-24.
［6］MCCLUSKEY B J，PELZEL-MCCLUSKEY A M，CREEKMORE L，et al.Vesicular stomatitis outbreak in the southwestern United States，2012［J］.J Vet Diagn Invest，2013，25(5)：608-613.
［7］MARTINEZ I，BARRERA J C，RODRIGUEZ L L，et al.Recombinant vesicular stomatitis(Indiana) virus expressing New Jersey and Indiana glycoproteins induces neutralizing antibodies to each serotype in swine，a natural host［J］.Vaccine，2004，22(29-30)：4035-4043.
［8］周学章，李元刚，高丰.水泡性口炎病毒保护性抗原基因核酸疫苗质粒的构建及其免疫原性［J］.中国生物制品学杂志，2008，21(6)：500-503，509.
ZHOU X Z，LI Y G，GAO F.Construction and immunogenictiy of recombinant DNA vaccine plasmid with protective antigen gene of vesicular stomatitis virus［J］. Chinese Journal of Biologicals，2008，21(6)：500-503，509.(in Chinese)
［9］PATTERSON L J，KUATE S，DALTABUIT-TEST M，et al.Replicating adenovirus-simian immunodeficiency virus(SIV) vectors efficiently prime SIV-specific systemic and mucosal immune responses by targeting myeloid dendritic cells and persisting in rectal macrophages，regardless of immunization route［J］.Clin Vaccine Immunol，2012，19(5)：629-637.
［10］DEAL C，PEKOSZ A，KETNER G.Prospects for oral replicating adenovirus-vectored vaccines［J］.Vaccine，2013，31(32)：3236-3243.
［11］SMALL J C，ERTL H C.Viruses—from pathogens to vaccine carriers［J］.Curr Opin Virol， 2011，1(4)：241-245.
［12］MONTEIL M，LE POTTIER M F，RISTOV A A，et al.Single inoculation of replication-defective adenovirus-vectored vaccines at birth in piglets with maternal antibodies induces high level of antibodies and protection against pseudorabies［J］.Vaccine， 2000，18(17)：1738-1742.
［13］WANG Y，LIU G，CHEN Z，et al.Recombinant adenovirus expressing F and H fusion proteins of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in goats［J］.Vet Immunol Immunopathol， 2013，154(1-2)：1-7.
［14］MAYR G A，O′DONNELL V，CHINSANGARAM J，et al.Immune responses and protection against foot-and-mouth disease virus(FMDV) challenge in swine vaccinated with adenovirus-FMDV constructs［J］.Vaccine， 2001，19(15-16)：2152-2162.
［15］MATHIEU M E，GRIGERA P R，HELENIUS A，et al.Folding，unfolding，and refolding of the vesicular stomatitis virus glycoprotein［J］.Biochemistry，1996，35(13)：4084-4093.
［16］SUN C，ZHAO K，CHEN K，et al.Development of a convenient immunochromatographic strip for the diagnosis of vesicular stomatitis virus serotype Indiana infections［J］.J Virol Methods，2013，188(1-2)：57-63.
［17］CASIMIRO D R，CHEN L，FU T M，et al.Comparative immunogenicity in rhesus monkeys of DNA plasmid，recombinant vaccinia virus，and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene［J］.J Virol，2003，77(11)：6305-6313.
［18］于龙，李劲松.重组腺病毒载体疫苗黏膜免疫机制与途径研究［J］.微生物学免疫学进展，2007，35(3)：69-73.
YU L，LI J S.Study onImmune mechanisms and pathways of recombinant adenoviral vector vaccine［J］. Progress in Microbiology and Immunology， 2007，35(3)：69-73.(in Chinese)
［19］张志晓，郑颖，李琦涵，等.中和抗体和细胞免疫在病毒疫苗有效性评价中的相互关系［J］.中国生物制品学杂志，2015，28(1)：91-94.
ZHANG Z X，ZHENG Y，LI Q H，et al.Relationship between neutralizing antibody and cellular immunity in effect evaluation of viral vaccines［J］. Chinese Journal of Biologicals，2015，28(1)：91-94.(in Chinese)

[1]	陈祥兴, 李蛟龙, 邢通, 张林, 高峰. 过氧化氢对成肌细胞的氧化损伤作用研究[J]. 畜牧兽医学报, 2019, 50(5): 1016-1025.
[2]	陈祥兴, 李蛟龙, 邢通, 张林, 高峰. 过氧化氢对成肌细胞的氧化损伤作用研究[J]. 畜牧兽医学报, 2019, 50(5): 1016-1025.
[3]	吴晋强, 曹校瑞, 闫瑞琴, 陆娜, 张娇娇, 吴佳豪, 赫晓燕. FGF21及其受体FGFR1和FGFR2在小鼠毛囊第1生长周期的表达[J]. 畜牧兽医学报, 2019, 50(3): 534-543.
[4]	吴晋强, 曹校瑞, 闫瑞琴, 陆娜, 张娇娇, 吴佳豪, 赫晓燕. FGF21及其受体FGFR1和FGFR2在小鼠毛囊第1生长周期的表达[J]. 畜牧兽医学报, 2019, 50(3): 534-543.
[5]	杨显英, 熊显荣, 韩杰, 黄向月, 王艳, 阿果约达, 李键. 小鼠卵巢组织定量PCR分析中内参基因的筛选[J]. 畜牧兽医学报, 2019, 50(2): 446-453.
[6]	杨显英, 熊显荣, 韩杰, 黄向月, 王艳, 阿果约达, 李键. 小鼠卵巢组织定量PCR分析中内参基因的筛选[J]. 畜牧兽医学报, 2019, 50(2): 446-453.
[7]	陈晓, 葛雷, 戴建军, 李丽, 张德福, 吴彩凤, 张树山. 羊口疮病毒F1L、B2L基因重组腺病毒构建及对小鼠的免疫效果分析[J]. 畜牧兽医学报, 2018, 49(8): 1701-1708.
[8]	黄伟宽, 刘瑞菊, 贾宁, 方梅, 陶波, 张嘉男, 梁鹏飞, 邓康. 沙冬青种子总黄酮对免疫抑制小鼠免疫功能及胸腺和脾超微结构的影响[J]. 畜牧兽医学报, 2018, 49(8): 1752-1760.
[9]	陈晓, 葛雷, 戴建军, 李丽, 张德福, 吴彩凤, 张树山. 羊口疮病毒F1L、B2L基因重组腺病毒构建及对小鼠的免疫效果分析[J]. 畜牧兽医学报, 2018, 49(8): 1701-1708.
[10]	黄伟宽, 刘瑞菊, 贾宁, 方梅, 陶波, 张嘉男, 梁鹏飞, 邓康. 沙冬青种子总黄酮对免疫抑制小鼠免疫功能及胸腺和脾超微结构的影响[J]. 畜牧兽医学报, 2018, 49(8): 1752-1760.
[11]	任万平, 邵伟, 雒诚龙, 余雄. 外源性添加磷酸二羟基丙酮对小鼠脂肪细胞三酰甘油及关键代谢酶含量的影响[J]. 畜牧兽医学报, 2018, 49(7): 1423-1431.
[12]	任万平, 邵伟, 雒诚龙, 余雄. 外源性添加磷酸二羟基丙酮对小鼠脂肪细胞三酰甘油及关键代谢酶含量的影响[J]. 畜牧兽医学报, 2018, 49(7): 1423-1431.
[13]	张建斌, 刘颖, 白立景, 牟玉莲, 韩俊文. 可控表达pGH基因转基因小鼠模型的建立与分析[J]. 畜牧兽医学报, 2018, 49(6): 1145-1153.
[14]	张建斌, 刘颖, 白立景, 牟玉莲, 韩俊文. 可控表达pGH基因转基因小鼠模型的建立与分析[J]. 畜牧兽医学报, 2018, 49(6): 1145-1153.
[15]	牛姝, 程笳琪, 高淑媛, 曹校瑞, 吴晋强, 陆娜, 闫瑞琴, 赫晓燕. FGF7亚家族在小鼠毛囊第一生长周期的表达[J]. 畜牧兽医学报, 2018, 49(3): 515-524.

表达水泡性口炎病毒双血清型G蛋白重组腺病毒的构建及对小鼠的免疫原性分析

Construction and Immunogenicity of Recombinant Adenovirus Expressing Two Serotypes of Vesicular Stomatitis Virus G Proteins in Mice

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