重组牛疱疹病毒1型转移载体的构建与初步应用

doi:10.3969/j.issn.1004-1524.20221496

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (10): 2311-2320.DOI: 10.3969/j.issn.1004-1524.20221496

重组牛疱疹病毒1型转移载体的构建与初步应用

戴莎莎(), 马小静, 王景松, 田兴苗, 王健霖, 李继东()

宁夏大学动物科技学院,宁夏银川,750021

收稿日期:2022-10-26 出版日期:2023-10-25 发布日期:2023-10-31
作者简介:戴莎莎(1995—),女,宁夏银川人,硕士研究生,研究方向为动物疫病诊断与防治。E-mail:1421164321@qq.com
通讯作者: ^*李继东,E-mail:lijidongi@foxmail.com
基金资助:
宁夏自然科学基金(2022AAC03075);宁夏回族自治区重点研发计划(2019ZWSF3007);宁夏回族自治区重点研发计划(2022BBF03024)

Construction and preliminary application of the universal transfer vector in recombination of Bovine herpes virus type 1

DAI Shasha(), MA Xiaojing, WANG Jingsong, TIAN Xingmiao, WANG Jianlin, LI Jidong()

College of Animal Science, Ningxia University, Yinchuan 750021, China

Received:2022-10-26 Online:2023-10-25 Published:2023-10-31

摘要/Abstract

摘要：

为构建一种能用于牛疱疹病毒1型(BHV-1)重组的转移载体,在真核表达载体pVAX1 CMV(cytomegalovirus)启动子的反向位点插入另一个CMV启动子,构建具有双向启动功能的表达载体prPP,在正、反向CMV启动子下游预置多克隆酶切位点;将绿色荧光蛋白标记基因置于反向启动子下,构建BHV-1重组载体prPgP;以BHV-1囊膜糖蛋白基因gE作为重组位点,将gE上下游同源臂插入prPgP载体中,牛病毒性腹泻病毒(BVDV)E2基因置于正向CMV启动子下,构建p△gErPgP-E2载体,用重组质粒转染已感染BHV-1的牛肾细胞(MDBK),产生的重组病毒经筛选、纯化、鉴定,命名为rBHV-1-△gE/E2,所含GFP和E2基因正常表达。结果表明,重组载体prPgP能方便地用于BHV-1的重组,为相关研究及其疫苗研制提供了便捷。

关键词: 牛疱疹病毒1型, 同源臂, 转移载体, 同源重组

Abstract:

The study aimed to construct a universal transfer vector that could be used for recombination of Bovine herpesvirus type 1 (BHV-1). The vector prPP with bidirectional promoter and two multiple cloning sites was constructed by insertion of another CMV promoter at the reverse site of the pVAX1 CMV promoter. The universal transfer vector prPgP was constructed by putting the green fluorescent protein marker gene downstream the reverse CMV promoter. Using the BHV-1 glycoprotein gE as the recombination site, the gE upstream and downstream homology arm was inserted into two multiple cloning sites of the prPgP vector respectively. Following, the recombinant transfer vector p△gErPgP-E2 was constructed by placing the Bovine viral diarrhea virus(BVDV) E2 gene as exogenous gene under the forward CMV promoter. The plasmid p△gErPgP-E2 was transfected with Madin-Darby Bovine kidney (MDBK) infected with BHV-1 to generate recombinant viruses. The recombinant viruses were screened, purified, identified, and named as rBHV-1-△gE/E2. The report gene GFP and BVDV E2 was expressed normally by fluorescence microscopy. The results showed that the recombinant universal vector prPgP could be convenient for BHV-1 recombination and be used in related research and its vaccine development.

Key words: Bovine herpesvirus type 1, homologous arm, transfer vector, homologous recombination

中图分类号:

S855.3

戴莎莎, 马小静, 王景松, 田兴苗, 王健霖, 李继东. 重组牛疱疹病毒1型转移载体的构建与初步应用[J]. 浙江农业学报, 2023, 35(10): 2311-2320.

DAI Shasha, MA Xiaojing, WANG Jingsong, TIAN Xingmiao, WANG Jianlin, LI Jidong. Construction and preliminary application of the universal transfer vector in recombination of Bovine herpes virus type 1[J]. Acta Agriculturae Zhejiangensis, 2023, 35(10): 2311-2320.

图/表 15

图1 载体prPgP构建示意图 prPP 多克隆位点(MCS)Ⅰ:SalⅠ, NsiⅠ, BglⅡ, EcoRⅠ, PstⅠ, EcoR V, NotⅠ, XhoⅠ, XbaⅠ, ApaⅠ; prPP 多克隆位点(MCS)Ⅱ:ClaⅠ, SaIⅠ, BspEⅠ, BamHⅠ, KpnⅠ, HindⅢ, AflⅡ, PmeⅠ, NheⅠ, XmaⅠ。

Fig.1 Schematic diagram of construction of universal vector prPgP prPP multiple cloning site (MCS) Ⅰ: SalⅠ, NsiⅠ, BglⅡ, EcoRⅠ, PstⅠ, EcoR V, NotⅠ, XhoⅠ, XbaⅠ, ApaⅠ; prPP multiple cloning site (MCS) Ⅱ: ClaⅠ, SaIⅠ, BspEⅠ, BamHⅠ, KpnⅠ, HindⅢ, AflⅡ, PmeⅠ, NheⅠ, XmaⅠ.

表1 prPgP载体构建引物序列与酶切位点

Table 1 Primer sequence and restriction site of prPgP vector

目的片段 Target fragment	引物序列 Primer sequence (5'→3')	酶切位点 Enzymatic cleavage site	退火温度 Annealing temperature/℃	长度 Length/bp
pVAX1-1	F: ATGCATAGATCTGAATTCTGCAGATATCCAG	NsiⅠ	55	2 966
	R: ATGCATCCGCGGCAGCTTGGGTCTCC	NsiⅠ
rCMV	F: AAAGTCGACTCCGGAGGATCCGAGCTCGGTAC	SalⅠ	60	629
	R: AAACCCGGGAATGGCCCGCCTGGCTG	XmaⅠ
pVAX1-2	F: TTTCCCGGGGACTCTTCGCGATGTAC	XmaⅠ	60	2 984
	R: AAAGTCGACATCGATAAGAACATGTGAGCAAAAG	SalⅠ
GFP	F: GGGCTTAAGTCACTTGTACAGCTCATCCA	AflⅡ	62	738
	R: AAAGCTAGCATGGTGAGCAAGGGCG	NheⅠ

表2 p△gErPgP-E2质粒引物序列与酶切位点

Table 2 p△gErPgP-E2 plasmid primer sequence and restriction site

目的片段 Target fragment	引物序列 Primer sequence (5'→3')	酶切位点 Enzymatic cleavage site	退火温度/℃ Annealing temperature	长度 Length/bp
gE L	F: AAGGATCCCGCCGGGTTGTTAAATG	BamHⅠ	66	654
	R: GGAAGCTTGATGAGCCGGTCGTACA	HindⅢ
gE R	F: AACTGCAGCCACGTGGAAGCTTGGGA	PstⅠ	68	795
	R: TGATATCGCCTCATCAGCGCCTCGA	EcoRⅤ
E2	F: ATAAGATCTCGCCACCATGGTGCACTTGGATTGCAA	BglⅡ	60	1 156
	R: GCGCTGCAGCTACCCTAAGGCCTTCTGTTCTGATAA	PstⅠ

图2 重组载体p△gErPgP-E2构建示意图

Fig.2 Schematic diagram of construction of recombinant vector p△gErPgP-E2

表3 重组病毒PCR鉴定引物序列

Table 3 Primer sequence for PCR identification of recombinant virus

目的片段 Target fragment	上游引物序列 Forward primer sequence (5'→3')	下游引物序列 Reverse primer sequence (5'→3')	退火温度 Annealing temperature/℃	长度 Length/bp
gE	ACTCAACGGGCGACAAAGAGTTT	TCTACGCTGTTATTGGCGGGAC	62	667
GFP	TTCACCTTGATGCCATTCTT	AGTGCTTCTCACGCTACCC	55	300
E2	CGCCACCATGGTGCACTTGGATTGCAA	CTACCCTAAGGCCTTCTGTTCTGATAA	60	1 144

图3 prPgP载体目的片段PCR产物电泳图 M1,DL5000 DNA分子量标准;1,pVAX1-1;M2,DL2000 DNA分子量标准;2,rCMV;3,pVAX1-2;M3,DL1000 DNA分子量标准;4,绿色荧光蛋白基因。

Fig.3 Electrophoresis of PCR products of PRPGP vector target fragment M1, DL5000 DNA marker; 1, pVAX1-1; M2, DL2000 DNA marker; 2, rCMV; 3, pVAX1-2; M3, DL1000 DNA marker; 4, GFP gene.

图4 prPP、prPgP重组质粒双酶切鉴定 M,DL5000 DNA分子量标准;1,prPP用SalⅠ、XmaⅠ双酶切;2,rCMV;3,pVAX1-1;4,prPgP用AflⅡ、NheⅠ双酶切;5,GFP基因;6,prPP。

Fig.4 Identification of recombinant plasmid prPP and prPgP by double restriction enzyme digestion M, DL5000 DNA marker; 1, Enzymatic cleavage of PrPP with SalⅠ and XmaⅠ; 2, rCMV; 3, pVAX1-1; 4, Enzymatic cleavage of prPgP with AflⅡ and NheⅠ; 5: GFP gene; 6: prPP.

图5 p△gErPgP-E2质粒目的片段PCR产物电泳图 M1,DL2000 DNA分子量标准;1,gE L片段;M2,DL1000 DNA分子量标准;2,gE R;3,E2基因。

Fig.5 Electrophoresis of PCR product of p△gErPgP-E2 plasmid target fragment M1, DL2000 DNA marker; 1, gE L; M2, DL1000 DNA marker; 2, gE R; 3, E2 gene.

图6 p△gErPgP-E2质粒双酶切鉴定 M,DL5000 DNA分子量标准;1,p△gELrPgP用BamHⅠ、HindⅢ双酶切;2,p△gELrPgP;3,rCMV;4,p△gErPgP用PstⅠ、EcoRⅤ双酶切;5,p△gErPgP;6,gE R片段;7,p△gErPgP-E2用BglⅡ、PstⅠ双酶切;8,p△gErPgP;9,BVDV E2。

Fig.6 Identification of p△gErPgP-E2 plasmid by double restriction enzyme digestion M, DL5000 DNA marker; 1, Enzymatic cleavage of p△gELrPgP with BamHⅠ and HindⅢ; 2, p△gELrPgP; 3, rCMV; 4, Enzymatic cleavage of p△gErPgP with PstⅠ and EcoRⅤ; 5, p△gELrPgP; 6, gE R; 7, Enzymatic cleavage of p△gErPgP-E2 with BglⅡ and PstⅠ; 8, p△gErPgP; 9, BVDV E2.

图7 重组病毒筛选过程中绿色荧光蛋白表达情况(100×) A、C、E是荧光条件下第1代、第4代、第9代BHV-1重组病毒在细胞培养中的绿色荧光变化情况;B、D、F分别是A、C、E对应的明场下细胞形态。

Fig.7 The expression of GFP in the process of recombinant virus screening (100×) A, C and E are the changes of green fluorescence of the 1st, 4th and 9th generation BHV-1 recombinant viruses in cell culture under fluorescent conditions; B, D and F are the cell morphology under bright field corresponding to A, C and E, respectively.

图8 重组病毒PCR鉴定 M1,DL500 DNA分子量标准;1,重组病毒GFP鉴定;2,GFP阳性对照;3,GFP阴性对照;M2,DL2000 DNA分子量标准;4,重组病毒缺失gE片段鉴定;5,gE阴性对照;6,gE阳性对照;7、8,重组病毒E2鉴定;9、10,E2阳性对照;11,E2阴性对照。

Fig.8 PCR identification of recombinant virus M1, DL500 DNA marker; 1, Identification of GFP in recombinant virus; 2, GFP positive control; 3, GFP negative control; M2, DL2000 DNA marker; 4, Deletion identification of gE fragment in recombinant virus; 5, gE positive control; 6, gE negative control; 7 and 8, Identification of E2 in recombinantvirus; 9 and 10, E2 positive control; 11, E2 negative control.

表4 BHV-1的TCID50测定结果

Table 4 Determination result of TCID50 of BHV-1

稀释倍数 Dilution multiple	病变数/总数 Lesion number/Total number	阳性占比 Positive ratio/%
10^-3	8/8	100
10^-4	8/8	100
10^-5	7/8	91.70
10^-6	4/8	50
10^-7	0/8	0
10^-8	0/8	0
阴性对照	0/8	0
Negative control

表5 rBHV-1-△gE/E2的TCID50测定结果

Table 5 Determination result of TCID50 of recombinant virus rBHV-1-△gE/E2 TCID50

稀释倍数 Dilution multiple	病变数/总数 Lesion number/Total number	阳性占比 Positive ratio/%
10^-3	8/8	100
10^-4	8/8	100
10^-5	6/8	75
10^-6	2/8	25
10^-7	0/8	0
10^-8	0/8	0
阴性对照	0/8	0
Negative control

图9 重组病毒与亲本病毒的生长曲线

Fig.9 Growth curve of recombinant virus and parent virus

图10 重组病毒接种MDBK细胞IFA结果(100×) A,重组毒株rBHV-1-ΔgE/E2感染细胞的荧光情况;C,亲本病毒BHV-1感染细胞的荧光情况;B、D分别是A、C对应的明场下细胞形态。

Fig.10 IFA result of MDBK cell inoculated with recombinant virus (100×) A is the fluorescence of recombinant strain rBHV-1-ΔgE/E2 infected cells; C is the fluorescence of parental virus BHV-1 infected cells; B and D are the cell morphology under bright field corresponding to A and C, respectively.

参考文献 15

[1]	HOU P L, ZHAO M, HE W Q, et al. Cellular microRNA bta-miR-2361 inhibits bovine herpesvirus 1 replication by directly targeting EGR1 gene[J]. Veterinary Microbiology, 2019, 233: 174-183.
[2]	杨彩凤. 肉牛传染性鼻气管炎的流行病学、临床症状、诊断及防控[J]. 现代畜牧科技, 2021(6): 88-89.
	YANG C F. Epidemiology, clinical symptoms, diagnosis, prevention and control of infectious rhinotracheitis in beef cattle[J]. Modern Animal Husbandry Science & Technology, 2021(6): 88-89. (in Chinese)
[3]	李东丽, 葛桂阳, 刘艺, 等. 牛传染性鼻气管炎疫苗的研究进展[J]. 中国兽医学报, 2021, 41(2): 360-365.
	LI D L, GE G Y, LIU Y, et al. Research progress on infectious bovine rhinotracheitis vaccine[J]. Chinese Journal of Veterinary Science, 2021, 41(2): 360-365. (in Chinese with English abstract)
[4]	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, 40(5): 102-110.
	XIE Q M, FENG K Y, SHEN Y. Advances in recombinant live vector vaccines for animal viruses[J]. Journal of South China Agricultural University, 2019, 40(5): 102-110. (in Chinese with English abstract)
[5]	王子书, 王萍, 许健, 等. 共表达鸡传染性法氏囊病病毒LX株VP2基因及鸡新城疫病毒PX02/3株F基因的重组火鸡疱疹病毒的构建[J]. 中国兽医杂志, 2020, 56(11): 26-30.
	WANG Z S, WANG P, XU J, et al. Construction of recombinant turkey herpes virus co-expressing infectious bursal disease virus LX strain VP2 gene and chicken newcastle disease virus PX02/3 strain F gene[J]. Chinese Journal of Veterinary Medicine, 2020, 56(11): 26-30. (in Chinese with English abstract)
[6]	FENG Z H, CHEN J H, LIANG W W, et al. The recombinant pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice[J]. Virology Journal, 2020, 17(1): 180.
[7]	SHAO Y H, SUN J F, HAN Z X, et al. Recombinant infectious laryngotracheitis virus expressing newcastle disease virus F protein protects chickens against infectious laryngotracheitis virus and Newcastle disease virus challenge[J]. Vaccine, 2018, 36(52): 7975-7986.
[8]	ALIGHOLIPOUR FARZANI T, FÖLDES K, HANIFEHNEZHAD A, et al. Bovine herpesvirus type 4 (BoHV-4) vector delivering nucleocapsid protein of crimean-congo hemorrhagic fever virus induces comparable protective immunity against lethal challenge in IFNα/β/γR-/-mice models[J]. Viruses, 2019, 11(3): 237.
[9]	郭巍, 朱远茂, 薛飞, 等. 牛疱疹病毒1型作为活病毒载体的研究进展[J]. 中国生物工程杂志, 2005, 25(S1): 32-35.
	GUO W, ZHU Y M, XUE F, et al. Progress on researches of bovine herpesvirus 1 as a living virus vector[J]. Progress in Biotechnology, 2005, 25(S1): 32-35. (in Chinese with English abstract)
[10]	杨明珠, 陈晓春, 宋佳诚, 等. 重组动物疱疹病毒活载体疫苗的构建与应用研究进展[J]. 中国畜牧兽医, 2022, 49(1): 338-351.
	YANG M Z, CHEN X C, SONG J C, et al. Research progress on construction and application of recombinant animal herpesvirus live vector vaccine[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(1): 338-351. (in Chinese with English abstract)
[11]	何小莉. PRV变异株GZDZ2016的分离鉴定与TK基因缺失转移载体的构建[D]. 贵阳: 贵州大学, 2019.
	HE X L. Isolation and identification of PRV mutant GZDZ2016 and construction of TK gene deletion transfer vector[D]. Guiyang: Guizhou University, 2019. (in Chinese with English abstract)
[12]	黄红亮, 何玲, 任常宝, 等. 独立表达双基因的伪狂犬病毒通用转移载体的构建[J]. 黑龙江畜牧兽医, 2016(19): 175-177.
	HUANG H L, HE L, REN C B, et al. Construction of universal transfer vector for pseudorabies virus expressing two genes independently[J]. Heilongjiang Animal Science and Veterinary Medicine, 2016(19): 175-177. (in Chinese)
[13]	王银. 表达牛病毒性腹泻病毒E2基因的重组I型牛疱疹病毒的构建[D]. 扬州: 扬州大学, 2013.
	WANG Y. Construction of recombinant bovine Herpes virus type I expressing E2 gene of bovine viral diarrhea virus[D]. Yangzhou: Yangzhou University, 2013. (in Chinese with English abstract)
[14]	冯军科, 朱远茂, 任宪刚, 等. 表达牛呼吸道合胞体病毒G蛋白基因的重组Ⅰ型牛疱疹病毒的构建与鉴定[J]. 中国预防兽医学报, 2010, 32(2): 81-85.
	FENG J K, ZHU Y M, REN X G, et al. Construction and identification of a recombinant BHV-1 expressing the G protein gene of bovine respiratory syncytial virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2010, 32(2): 81-85. (in Chinese with English abstract)
[15]	任宪刚, 薛飞, 朱远茂, 等. 表达O型口蹄疫病毒VP1基因的重组病毒BHV-1的构建与鉴定[J]. 微生物学报, 2009, 49(5): 678-683.
	REN X G, XUE F, ZHU Y M, et al. Construction and identification of recombinant virus BHV-1 expressing VP1 gene of foot-and-mouth disease virus O[J]. Acta Microbiologica Sinica, 2009, 49(5): 678-683. (in Chinese)

重组牛疱疹病毒1型转移载体的构建与初步应用

Construction and preliminary application of the universal transfer vector in recombination of Bovine herpes virus type 1

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 15

参考文献 15

相关文章 2

编辑推荐

Metrics

本文评价

[1]	李燕乐, 钟怀荣, 宣宁, 张燕, 陈高, 季祥. 磷酸泛酰巯基乙胺基转移酶基因过量表达对集胞藻PCC6803脂肪酸合成的影响[J]. 浙江农业学报, 2022, 34(6): 1316-1325.
[2]	路晓媛, 钟怀荣, 夏志洁, 曹月蕾, 陈高, 戴美学. 集胞藻酰基载体蛋白基因过量表达对脂肪酸合成的影响[J]. 浙江农业学报, 2020, 32(7): 1253-1262.