表达PRRSV NADC30-like毒株GP5-M的重组伪狂犬病病毒的构建及其生物学特性探究

doi:10.3969/j.issn.1004-1524.20221629

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (11): 2555-2567.DOI: 10.3969/j.issn.1004-1524.20221629

表达PRRSV NADC30-like毒株GP5-M的重组伪狂犬病病毒的构建及其生物学特性探究

孙珊珊¹(), 其美拉姆², 李强¹, 曾南方³, 郑诚⁴, 张白玉¹, 颜其贵¹^,^*()

1.四川农业大学动物医学院,四川成都 611130
2.林芝市察隅县上察隅镇农牧综合中心,西藏察隅 860614
3.巨星农牧有限公司,四川成都 611130
4.四川省丽天牧业有限公司,四川达州 635099

收稿日期:2022-11-16 出版日期:2023-11-25 发布日期:2023-12-04
作者简介:孙珊珊(1994—),女,河南开封人,硕士研究生,主要从事微生物与免疫研究。E-mail: 1281101296@qq.com
通讯作者: * 颜其贵,E-mail: yanqigui@126.com
基金资助:
成都市重大科技应用项目(2022-YF09-00050-SN)

Construction and biological characteristics of a recombinant pseudorabies virus expressing GP5-M of PRRSV NADC30-like virus strain

SUN Shanshan¹(), CHEMI Lhamo², LI Qiang¹, ZENG Nanfang³, ZHENG Cheng⁴, ZHANG Baiyu¹, YAN Qigui¹^,^*()

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Shangchayu Town Agricultural and Animal Husbandry Comprehensive Center, Zayü County, Zayü 860614, Xizang, China
3. Juxing Agriculture and Animal Husbandry Co., Ltd., Chengdu 611130, China
4. Sichuan Litian Animal Husbandry Co., Ltd., Dazhou 635099, Sichuan, China

Received:2022-11-16 Online:2023-11-25 Published:2023-12-04

摘要/Abstract

摘要：

为获得一株能够融合表达猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)GP5-M蛋白的重组伪狂犬病病毒PRV-GP5-M毒株,以猪伪狂犬病病毒(pseudorabies virus, PRV)变异株TK基因缺失株PRV FJ01/TK-为病毒载体,以gI/gE基因为插入靶点,利用同源重组技术和CRISPR/Cas9技术敲除gI/gE基因,并在gI/gE位置上插入CMV-GP5-M表达盒,经噬斑纯化,成功构建能够正确表达GP5-M蛋白重组病毒PRV-GP5-M。进一步对该毒株的稳定性、生长动力学、培养特性、安全性等生物学特性进行探究。结果表明,该重组毒株具有良好的遗传稳定性、安全性,易于增殖培养。研究结果为靶向PRRSV GP5与M蛋白生成新的PRRSV疫苗提供了新的线索,同时可为预防近些年流行的PRRSV NADC30-like毒株和PRV变异株的疫苗研发提供重要参考。

关键词: GP5-M蛋白, 重组伪狂犬病病毒, CRISPR/Cas9, 生物学特性

Abstract:

To obtain a recombinant pseudorabies virus PRV-GP5-M strain that can fuse and express the GP5-M protein of porcine reproductive and respiratory syndrome virus (PRRSV), using the porcine pseudorabies virus (PRV) variant TK gene deletion strain PRV FJ01/TK-as the viral vector and the gI/gE gene as the insertion target, the gI/gE gene was knocked out using homologous recombination technology and CRISPR/Cas9 technology, and a CMV-GP5-M expression box was inserted at the gI/gE position. After plaque purification, a recombinant virus PRV-GP5-M that could correctly express the GP5-M protein was successfully constructed. Furthermore, the biological characteristics of the strain, including stability, growth kinetics, culture characteristics and safety were explored. The results showed that the recombinant strain had good genetic stability, safety, and was easy to proliferate and cultivate. This study result provided new clues for targeting PRRSV GP5 and M protein to generate new PRRSV vaccines, and provided important references for vaccines against the prevalent PRRSV NADC30-like and PRV variant strains in recent years.

Key words: GP5-M protein, recombinant pseudorabies virus, CRISPR/Cas9, biological characteristics

中图分类号:

S855.3

孙珊珊, 其美拉姆, 李强, 曾南方, 郑诚, 张白玉, 颜其贵. 表达PRRSV NADC30-like毒株GP5-M的重组伪狂犬病病毒的构建及其生物学特性探究[J]. 浙江农业学报, 2023, 35(11): 2555-2567.

SUN Shanshan, CHEMI Lhamo, LI Qiang, ZENG Nanfang, ZHENG Cheng, ZHANG Baiyu, YAN Qigui. Construction and biological characteristics of a recombinant pseudorabies virus expressing GP5-M of PRRSV NADC30-like virus strain[J]. Acta Agriculturae Zhejiangensis, 2023, 35(11): 2555-2567.

图/表 17

表1 引物信息表

Table 1 Primers information

引物名称 Primer name	上游引物 Forward primer (5’→3’)	下游引物 Reverse primer (5’→3’)	产物长度 Product length/bp
gI/gE-Left	CCATGATTACGCCAAGCTTTTGCGTACGGCCTTGCTTACGGG	CCGTAATTGATTACTATTAATCGATGGaagcttctacggaccgggct	1 351
gI/gE-eGFP	agcccggtccgtagaagcttCCATCGATTAATAGTAATCAATTACGG	aaacgtgtccatgtcgaaggCCTCTTGTACAGCTCGTCCATGC	1 366
gI/gE-Right	GCATGGACGAGCTGTACAAGAGGccttcgacatggacacgttt	tgtaaaacgacggccagtgaattcGTGCCAGAGCGAGAGCGT	1 402
GP5-M	tgaaccgtcagatccgctagcGCCACCATGCTGGGCAAG	ACGTGTCCATGTCGAAGGCCTTTACTTGGCGTACTTCACCAGGT	1 221

图1 pUC19-gI/gE/GP5-M载体设计

Fig.1 The designs of pUC19-gI/gE/GP5-M vector

表2 SgRNA引物信息表

Table 2 The primers information of SgRNA

向导RNA SgRNA	上游引物Forward primer(5’→3’)	下游引物Reverse primer(5’→3’)
gI/gE-1	CACCGGTGCACCACGAAGCCTTCCGCGG	AAACCCGCGGAAGGCTTCGTGGTGCACC
gI/gE-2	CACCGGGACGAGTTCAGCAGCGACGAGG	AAACCCTCGTCGCTGCTGAACTCGTCCC

图2 病毒构建示意图

Fig.2 Schematic diagram of constructing recombinant strain

表3 动物实验方案

Table 3 Animal experimental scheme

分组 Group		数量 Number	剂量 Dosage/(TCID₅₀·mL^-1)
A(PRV-GP5-M)	A-1	6	10⁴
	A-2	6	10⁵
	A-3	6	10⁶
B(PRV FJ01)	B-1	6	10⁴
	B-2	6	10⁵
	B-3	6	10⁶
C(DMEM)		6	100 μL

图3 gI/gE转移载体片段扩增 A,M为DL2000 DNA分子量标准,1、2、3分别为片段gI/gE-Left、gI/gE-eGFP、gI/gE-Right。B,M为DL2000分子量标准,1为GP5-M片段。

Fig.3 PCR amplification of gI/gE transfer vector A, M was DL2000 DNA marker, 1 was gI/gE-Left, 2 was gI/gE-eGFP, 3 was gI/gE-Right. B, M was DL2000 marker, 1 was GP5-M.

图4 Western blot鉴定转移载体中GP5-M蛋白的表达 M为蛋白分子量标准;1为转移载体pUC19-gI/gE/GP5-M。

Fig.4 Western blot analysis of the GP5-M protein expressed by pUC19-gI/gE/GP5-M M, Protein marker; 1, Transfer vector pUC19-gI/gE/GP5-M.

图5 SgRNA载体构建鉴定结果 M为DL2000 DNA分子量标准,1、2分别为pX459-gIgE-SgRNA1、pX459-gIgE-SgRNA2的SgRNA鉴定结果。

Fig.5 Detection results of SgRNA vectors M, DL2000 DNA marker,1 and 2 were pX459-gIgE-SgRNA1 and pX459-gIgE-SgRNA2,respectively.

图6 SgRNA载体测序结果

Fig.6 Sequence information of SgRNA vector

图7 PRV-GP5-M鉴定结果 M为DL5000 DNA分子量标准;1、2分别为PRV PRV FJ01、PRV-GP5-M的gI/gE鉴定引物PCR结果;3为PRV-GP5-M的GP5-M基因引物鉴定结果。

Fig.7 Detection results of PRV-GP5-M M, DL5000 DNA marker; 1, PCR result of gI/gE identification primers of PRV FJ01; 2, PCR result of gI/gE identification primers of PRV-GP5-M; 3, Identification result of GP5-M gene primers of PRV-GP5-M.

图8 Western blot鉴定重组病毒中GP5-M蛋白的表达 M为蛋白分子量标准;1、2分别为PRV-GP5-M、PRV FJ01。

Fig.8 Western blot analysis of the GP5-M protein expressed by PRV-GP5-M M, Protein marker; 1, PRV-GP5-M; 2, PRV FJ01.

图9 重组病毒感染PK15细胞后间接免疫荧光检测GP5-M蛋白的表达

Fig.9 IFA identification of GP5-M protein expressed by PRV-GP5-M A, PRV-GP5-M; B, PRV FJ01.

图10 重组病毒的遗传稳定性 A,PCR检测重组病毒PRV-GP5-M的遗传稳定性;M1为DL 2000 DNA分子量标准,F5、F10、F15分别为重组病毒PRV-GP5-M第5、10、15代GP5-M基因检测结果。B,第5、10、15代中GP5-M基因同源性比对结果。C,Western blot检测F5、F10、F15中GP5-M蛋白的表达;M2为蛋白分子量标准,F5、F10、F15分别为重组病毒PRV-GP5-M第5、10、15代中GP5-M蛋白表达结果。

Fig.10 Genetic stability of recombinant virus A, PCR detection for genetic stability of recombinant virus; M1, DL 2000 DNA maker; F5, F10 and F15 were the GP5-M gene detection results of the 5th, 10th and 15th generations of the recombinant virus PRV-GP5-M, respectively. B, Homology comparison of GP5-M gene in the 5th, 10th and 15th generation. C, The expression of GP5-M protein in recombinant virus PRV-GP5-M was detected by Western blot; M2, Protein marker, F5, F10, F15 were the expression results of the 5th, 10th, 15th generation GP5-M protein in the recombinant virus PRV-GP5-M.

图11 一步生长曲线的测定

Fig.11 One-step growth curve of PRV-GP5-M and PRV FJ01 strain on PK15

图12 重组病毒在不同细胞中的培养特性(20×) A、B、C、D、E、F分别为重组病毒PRV-GP5-M与PRV FJ01在IPEC、Marc145、MDCK、PK15、ST、Vero上的培养特性。

Fig.12 Culture characteristics of recombinant virus in different cells (20×) A, B, C, D, E and F were the culture characteristics of recombinant virus PRV-GP5-M and PRV FJ01 on IPEC, Marc145, MDCK, PK15, ST and Vero, respectively.

图13 两株病毒在不同细胞上的TCID50 ****,P <0.001;NS,不显著。下同。

Fig.13 TCID50 of recombinant viruses PRV-GP5-M and PRV FJ01 on different cells ****, P <0.001; NS, Not significant. The same as below.

图14 不同器官中的病毒载量 A,荧光定量PCR标准曲线;B,荧光定量PCR熔解曲线;C,不同器官中病毒核酸拷贝数,****, P<0.001。

Fig.14 Viral load in different organs A, Standard curve of fluorescent quantitative PCR. B, Melting curve of fluorescent quantitative PCR. C, Virus nucleic acid copy number in different organs, **** indicated P<0.001.

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表达PRRSV NADC30-like毒株GP5-M的重组伪狂犬病病毒的构建及其生物学特性探究

Construction and biological characteristics of a recombinant pseudorabies virus expressing GP5-M of PRRSV NADC30-like virus strain

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