含有不同启动子、WPRE以及ITRs对重组杆状病毒表达新城疫F蛋白的影响

doi:10.11924/j.issn.1000-6850.casb2020-0011

中国农学通报 ›› 2020, Vol. 36 ›› Issue (30): 91-97.doi: 10.11924/j.issn.1000-6850.casb2020-0011

所属专题：生物技术

含有不同启动子、WPRE以及ITRs对重组杆状病毒表达新城疫F蛋白的影响

曹德宸¹^,²(), 叶磊¹^,², 李维庆¹^,², 李赛男¹^,², 高冬妮¹^,²(), 葛菁萍¹^,²()

¹黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨 150500
²黑龙江大学生命科学学院黑龙江省普通高校微生物重点实验室,哈尔滨 150080

收稿日期:2019-04-16 修回日期:2020-05-24 出版日期:2020-10-25 发布日期:2020-10-16
通讯作者: 高冬妮,葛菁萍
作者简介:曹德宸,男,1994年出生,黑龙江牡丹江人,硕士研究生,研究方向：生物学。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号,Tel：0451-86609016,E-mail： 18845142771@163.com。
基金资助:
黑龙江省自然科学基金项目“鸡传染性法氏囊病病毒基因工程疫苗的研制、教育部留学回国人员基金(鸡新城疫疫苗的研制)”(D0249);黑龙江省青年创新人才培养计划(UNPYSCT-2016178);黑龙江省科学基金(C2017056);黑龙江省博士后资助项目(LBH-Z17191)

Effects of Different Promoters, WPRE and ITRs on Recombinant Baculovirus Expression of Newcastle Disease F Protein

Cao Dechen¹^,²(), Ye Lei¹^,², Li Weiqing¹^,², Li Sainan¹^,², Gao Dongni¹^,²(), Ge Jingping¹^,²()

¹Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500
²Key Laboratory of Microbiology, College of Heilongjiang Province, School of Life Sciences, Heilongjiang University, Harbin 150080

Received:2019-04-16 Revised:2020-05-24 Online:2020-10-25 Published:2020-10-16
Contact: Gao Dongni,Ge Jingping

摘要/Abstract

摘要：

为了提高基因工程疫苗对新城疫病毒F蛋白的表达能力,从而为研制高效基因工程疫苗奠定了基础。利用Western blot技术检测含有不同启动子与表达元件的重组杆状病毒,在中国仓鼠卵巢细胞中其F蛋白的表达量的差异。Western blot结果显示,含有CMV立即早期增强子区与鸡肌动蛋白启动子区的复合启动子的质粒表达F蛋白量较含人巨细胞病毒立即早期启动子(CMV)的质粒提高了24.52%~231.18%,含有土拨鼠肝炎病毒转录后调控元件(WPRE)能够提高F蛋白表达量2.9~8.6倍,而腺伴随病毒末端反向重复序列(ITRs)对质粒F蛋白表达量提高1.95%~9.27%。CBA启动子的启动能力远高于CMV的启动能力,调控元件WPRE能够显著提高F蛋白的表达量,而ITRs元件对于F蛋白的表达量未产生显著提高。

关键词: 新城疫病毒, 杆状病毒, 土拨鼠肝炎病毒转录后调控元件, 含有CMV立即早期增强子区与鸡肌动蛋白启动子区的复合启动子

Abstract:

The aims are to improve the expression ability of genetically engineered vaccines to the Newcastle disease virus F protein, and lay a foundation for developing highly efficient and genetically engineered vaccines. The expression differences of baculovirus with different promoters and expression elements to F protein in Chinese hamster ovary cell were detected by Western blot. The results of Western blot showed that the expression level of complex promoter containing CMV enhancer region and chicken actin promoter region (CBA promoter) was higher than that of Cytomegaoviyns promoter (CMV) by 24.52%-231.18%, and the expression level of wood-chuck hepatitis virus post-transcriptional regulatory element (WPRE) was increased by 2.9-8.6 times, but adeno-associated virus inverted terminal repeats (ITRs) only increased the expression level of 1.95%-9.27%. In conclusion, the adjustment ability of CBA promoter is much higher than that of CMV promoter, while group WPRE could get a positive result, but group ITRs does not change significantly.

Key words: Newcastle disease virus, baculovirus, WPRE, CBA

中图分类号:

S852.65

曹德宸, 叶磊, 李维庆, 李赛男, 高冬妮, 葛菁萍. 含有不同启动子、WPRE以及ITRs对重组杆状病毒表达新城疫F蛋白的影响[J]. 中国农学通报, 2020, 36(30): 91-97.

Cao Dechen, Ye Lei, Li Weiqing, Li Sainan, Gao Dongni, Ge Jingping. Effects of Different Promoters, WPRE and ITRs on Recombinant Baculovirus Expression of Newcastle Disease F Protein[J]. Chinese Agricultural Science Bulletin, 2020, 36(30): 91-97.

图/表 10

图1. pLM(-)、pLM、pLM-ITRs质粒图谱

图2. pSD(-)、pSD、pSD-ITRs质粒图谱

连接反应体系成分	体积
10×ligation Buffer	1 μL
目的基因片段	4 μL (40 ng)
线性质粒载体	1 μL (10 ng)
T4 DNA Ligase	1 μL
ddH₂O	Up to 10 μL

表1. 连接反应体系

图3. Xba Ⅰ/EcoR Ⅰ酶切鉴定 M：DNA Marker DL15000;泳道1：pYL-LM(-)(Xba I/EcoR I)双酶结果;泳道2：pYL-SD(-)(Xba I/EcoR I)双酶结果;泳道3：pYL-pYL-LM(Xba I/EcoR I)双酶结果;泳道4：pYL-LM-ITRs(Xba I/EcoR I)双酶结果;泳道5：pYL-SD(Xba I/EcoR I)双酶结果;泳道6：pYL-SD-ITRs(Xba I/EcoR I)双酶结果

图4. Xba Ⅰ/EcoR Ⅰ双酶切模式图 A：Xba I/EcoR I双酶切pYL-LM模式图;B：Xba I/EcoR I双酶切pYL-LM-I模式图;C：Xba I/EcoR I双酶切pYL-SD模式图;D：Xba I/EcoR I双酶切pYL-SD-I模式图

图5. YL-up,YL-down为引物PCR鉴定Bacmid M1：DNA Marker DL2000;M2：DNA Marker DL15000;泳道1：bYL-LM PCR鉴定结果;泳道2：bYL-LM-ITRs PCR鉴定结果;泳道3：bYL-LM(-) PCR鉴定结果;泳道4：bYL-SDPCR鉴定结果;泳道5：bYL-SD-ITRs PCR鉴定结果;泳道6：bYL-SD(-) PCR鉴定结果

病毒名称	滴度/(pfu/mL)
vYL-LM	2.2×10⁸
vYL-LM-I	3.0×10⁸
vYL-SD	2.8×10⁸
vYL-SD-I	3.2×10⁸
vYL-LM(-)	2.3×10⁸
vYL-SD(-)	2.7×10⁸

表2. 重组杆状病毒最终滴度

图6. F蛋白的SDS-PAGE结果 M：蛋白质marker;泳道1：vYL-LM侵染CHO-K1细胞收获蛋白SDS-PAGE结果;泳道2：vYL-LM-ITRs侵染CHO-K1细胞收获蛋白SDS-PAGE结果;泳道3：vYL-LM(-)侵染CHO-K1细胞收获蛋白SDS-PAGE结果;泳道4：vYL-SD侵染CHO-K1细胞收获蛋白SDS-PAGE结果;泳道5：vYL-SD-ITRs侵染CHO-K1细胞收获蛋白SDS-PAGE结果;泳道6：vYL-SD(-)侵染CHO-K1细胞收获蛋白SDS-PAGE结果;泳道7：未用任何病毒感染空白对照SDS-PAGE结果

图7. F基因表达产物Western blot结果分析 M：蛋白质marker;泳道1：空细胞对照;泳道2：vYL-SD侵染CHO-K1细胞收获蛋白Western blot结果;泳道3：vYL-SD-ITRs侵染CHO-K1细胞收获蛋白Western blot结果;泳道4：vYL-SD(-)侵染CHO-K1细胞收获蛋白western blot结果;泳道5：vYL-LM侵染CHO-K1细胞收获蛋白Western blot结果;泳道6：vYL-LM-ITRs侵染CHO-K1细胞收获蛋白Western blot结果;泳道7：vYL-LM(-)侵染CHO-K1细胞收获蛋白Western blot结果

组一	分子量	强度	组二	分子量	强度
泳道2 vYL-SD	57.48	1109.7	泳道5 vYL-LM	55.661	831.52
泳道3 vYL-SD-ITRs	56.563	1015.6	泳道6 vYL-LMI	55.661	815.61
泳道4 vYL-SD(-)	56.563	285.69	泳道7 vYL-LM(-)	54.772	86.264
泳道1 空白细胞对照	0	0

表3. Western blot软件分析结果

参考文献 27

[1]	B.W.卡尔尼克. 禽病学[M].第十版. 北京: 中国农业出版社, 1999: 691-726.
[2]	Pitt J J, Da Silva EGerman J J. Determination of the disulfide bond arrangement of Newcastle disease virus hemaggulutinon neuraminidase[J]. J Bio Chem, 2000,275(9):6469-6478.
[3]	Palmieri S, Michael L P. An alternative method of oligonucleotide finger printing for resulting Newcastle disease virus specific RNA fragments[J]. Avian Disease, 1988,33:345-350. doi: 10.2307/1590854 URL
[4]	Peeters B P, De Leeuwenhoek O S, Koch G. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence[J]. J Virol, 1999,73(6):5001-5009. doi: 10.1128/JVI.73.6.5001-5009.1999 URL pmid: 10233962
[5]	田夫林, 陈静, 马惠玲, 等. 10株新城疫病毒分离F基因的克隆及遗传变异分析[J]. 中国预防兽医学报, 2004(01):31-34.
[6]	Leilei D, Pucheng C, Xingzhi B, et al. Recombinant duck enteritis viruses expressing the Newcastle disease virus (NDV) F gene protects chickens from lethal NDV challenge[J]. Vet Microbiol, 2019,232:146-150. doi: 10.1016/j.vetmic.2019.04.022 URL pmid: 31030839
[7]	刘长梅. 新城疫病毒的研究现状[J]. 山东家禽, 2002(3):22-24.
[8]	Ding L, Chen P, Bao X. Recombinant duck enteritis viruses expressing the Newcastle disease virus (NDV) F gene protects chickens from lethal NDV challenge[J]. Veterinary Microbiology, 2019,232:146-150. doi: 10.1016/j.vetmic.2019.04.022 URL pmid: 31030839
[9]	谢青梅, 封柯宇, 沈勇. 动物病毒重组活载体疫苗研究进展[J]. 华南农业大学学报, 2019(5).
[10]	Ebadat S, Ahmadi S, Ahmadi M, et al. Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells[J]. Plos One, 2017,12(10):e0185967. doi: 10.1371/journal.pone.0185967 URL pmid: 29023479
[11]	James G. Ackford, Juan C. Corredor, Yanlong Pei a. et al. Foreign gene expression and induction of antibody response by recombinant fowl adenovirus-9-based vectors with exogenous promoters[J]. Vaccine, 2017,35(37):4974-4982. URL pmid: 28780115
[12]	Makrides S C. Components of vectors for gene transfer and expression in mammalian cells[J]. Protein ExprPurif, 1999,(17):183-202.
[13]	Powell S K, Rivera-Soto R, Gray S J. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy[J]. Discovery medicine, 2015,19(102):49-57. URL pmid: 25636961
[14]	高冬妮, 平文祥, 金丽颖, 等. 以具有WPRE调控元件的杆状病毒为载体在鸡胚原代细胞中表达新城疫病毒F基因[J]. 微生物学报, 2014,54(4):455-462.
[15]	Lizheng W, Zixuan W, Fangfang Z, et al. Enhancing Transgene Expression from Recombinant AAV8 Vectors in Different Tissues Using Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element[J]. International Journal of Medical Sciences, 2016,13(4):286-291. URL pmid: 27076785
[16]	Savy A, Dickx Y, Nauwynck L. Impact of Inverted Terminal Repeat Integrity on rAAV8 Production Using the Baculovirus/Sf9 Cells System[J]. Human gene therapy methods, 2017. doi: 10.1089/hgtb.2018.211 URL pmid: 30727774
[17]	Want C Y, Li F, Yang Y. Recombinant baculoviruses containing the Diphtheria toxin a gene for malignant glioma therapy[J]. Cancer Research, 2006,66:5798-5806. doi: 10.1158/0008-5472.CAN-05-4514 URL pmid: 16740719
[18]	高冬妮, 金丽颖, 葛菁萍, 等. EF1α启动的高效重组杆状病毒载体的构建及其表达效果[J]. 微生物学报, 2014(6).
[19]	Zhou Q, Tian W, Liu C, et al. Deletion of the B-B’ and C-C’ regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression[J]. Scientific Reports, 2017,7(1):5432. doi: 10.1038/s41598-017-04054-4 URL pmid: 28710345
[20]	Bello M B, Yusoff K, Ideris A, et al. Diagnostic and Vaccination Approaches for Newcastle Disease Virus in Poultry: The Current and Emerging Perspectives[J]. Biomed Res Int., 2018,2018:7278459. doi: 10.1155/2018/7278459 URL pmid: 30175140
[21]	Absalón A E, Cortés-Espinosa D V, Lucio E, et al. Epidemiology, control, and prevention of Newcastle disease in endemic regions: Latin America[J]. Trop Anim Health Prod. 2019,51(5):1033-1048. doi: 10.1007/s11250-019-01843-z URL pmid: 30877525
[22]	Firouzamandi M, Moeini H, Hosseini D, et al. Improved immunogenicity of Newcastle disease virus fusion protein genes[J]. Journal of Veterinary Science, 2016,17(1):21-26. doi: 10.4142/jvs.2016.17.1.21 URL pmid: 27051336
[23]	Das A T, Tenenbaum L, Berkhout B. Tet-On Systems For Doxycycline-inducible Gene Expression[J]. Curr Gene Ther., 2016,16(3):156-67. doi: 10.2174/1566523216666160524144041 URL pmid: 27216914
[24]	Chen B D, He C H, Chen X C, et al. Targeting transgene to the heart and liver with AAV9 by different promoters[J]. Clinical and Experimental Pharmacology and Physiology, 2015,42(10):1108-1117. URL pmid: 26173818
[25]	Zufferey R, Donello J E, Trono D, et al. Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors[J]. Journal of Virology, 1999,73(4):2886-2892. doi: 10.1128/JVI.73.4.2886-2892.1999 URL pmid: 10074136
[26]	Hu Y C. Baculoviral Vectors for Gene Delivery: A Review. Current Gene Therapy, 2008(8):54-65.
[27]	Wang C Y, Li F, Yang Y, et al. Recombinant baculovirus containing the Diphtheria toxin a gene for malignant glioma therapy[J]. Cancer Research, 2006,66:5798-5806. doi: 10.1158/0008-5472.CAN-05-4514 URL pmid: 16740719

含有不同启动子、WPRE以及ITRs对重组杆状病毒表达新城疫F蛋白的影响

Effects of Different Promoters, WPRE and ITRs on Recombinant Baculovirus Expression of Newcastle Disease F Protein

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 27

相关文章 11

编辑推荐

Metrics

本文评价

关于本刊

作者中心

审者中心

在线期刊

联系我们

[1]	王贝贝,宋丽丽,马德慧,毛景东,王学理. 中药鱼腥草注射液鸡胚接种抗新城疫病毒的研究[J]. 中国农学通报, 2018, 34(2): 95-97.
[2]	于航,高冬妮,田兆峰,平文祥,葛菁萍. 不同启动子对HA-VP2重组杆状病毒蛋白表达的影响[J]. 中国农学通报, 2017, 33(23): 100-105.
[3]	葛菁萍,白程乐,高冬妮,申艳,于航,平文祥. 鸡IL-2重组杆状病毒的构建及其病理组织学研究[J]. 中国农学通报, 2017, 33(21): 142-148.
[4]	刘颖,金丽颖,申燕,高冬妮,平文祥,葛菁萍. 新城疫病毒La Sota株HN基因的克隆与序列分析[J]. 中国农学通报, 2015, 31(14): 89-95.
[5]	安琦申燕原韬刘颖金丽颖高冬妮葛菁萍平文祥. 鸡新城疫病毒F基因的克隆及序列分析[J]. 中国农学通报, 2014, 30(26): 11-16.
[6]	唐晓艳高冬妮李梅葛菁萍平文祥楼庄伟. 昆虫细胞中表达eGFP基因的重组杆状病毒的构建[J]. 中国农学通报, 2013, 29(8): 26-30.
[7]	王立新,路振香,王金虎,王珊珊,胡静,戴四发. 新城疫病毒诱导黄粉虫抗病毒肽的效果分析[J]. 中国农学通报, 2009, 25(6): 37-39.
[8]	王学兵,张红英,徐端红,崔保安,程晓静,李欣,马姣. 几种中药在鸡胚上对鸡常见病毒的作用效果试验[J]. 中国农学通报, 2009, 25(13): 5-9.
[9]	崔斓斓. 麻杏石甘汤中抗新城疫病毒有效成分的研究[J]. 中国农学通报, 2008, 24(10): 35-38.
[10]	詹爱军　王新卫　谭婉明　马静云　毕英佐　于康振　曹永长. H9N2亚型AIV HA基因的克隆及其在昆虫细胞中的表达[J]. 中国农学通报, 2008, 24(1): 18-22.
[11]	许信刚,张琦,邢福珊,张耀相. Development and Application of a Diagnostic Kit for Detecting NDV[J]. 中国农学通报, 2005, 21(4): 30-30.