猪传染性胃肠炎病毒 <i>S、N</i>基因DNA疫苗载体构建及其免疫原性

成伟伟; 容维中; 杨明; 李元新; 赵子惠; 陈伯祥; 王佳; 周瑶

猪传染性胃肠炎病毒 S、N基因DNA疫苗载体构建及其免疫原性

甘肃省畜牧兽医研究所，甘肃　平凉　744000

基金项目: 甘肃省青年科技基金计划项目（21JR7RA719）；甘肃省农业生物技术研究与应用开发项目（GNSW-2011-24）；甘肃省重点研发计划项目（18YF1NA021-1）

详细信息

作者简介:
成伟伟（1988 —），男，硕士，副研究员，主要从事动物病原生物学与免疫学研究，E-mail：549861054@qq.com

中图分类号: S855.3
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出版历程
- 收稿日期: 2024-06-03
- 修回日期: 2024-08-23
- 网络出版日期: 2024-10-29

Construction and Immunogenicity of S and N gene DNA Vaccine Vector for TGEV

Gansu Institute of Animal and Veterinary Science , Pingliang, Gansu　744000, China

摘要

摘要: 目的构建猪传染性胃肠炎病毒（transmissible gastroenteritis virus, TGEV）S、N基因的DNA疫苗载体，并进行免疫原性试验，为猪传染性胃肠炎（transmissible gastroenteritis, TGE）的防控和DNA疫苗研究提供技术支撑和基础数据。方法扩增S基因的A位点、D位点和N基因，并将N基因（单独）、A位点和D位点（融合）克隆至pCDNA3.1-His-C构建重组疫苗载体，运用生物信息学软件预测分析S（A-D）蛋白、N蛋白二级结构组成、三级构像、亚细胞定位和优势B细胞抗原表位。将构建成功的重组载体分别转染至PK-15细胞进行间接免疫荧光试验，运用共聚焦检测重组蛋白的表达分布情况。将重组疫苗载体单独或联合免疫小鼠，运用间接ELISA检测IgG抗体水平。结果扩增出S基因的A位点、D位点和N基因，大小分别为498、606、1149 bp。构建了A位点与D位点（融合）、N基因（单独）的DNA疫苗重组载体p-S（A-D）-His和p-N-His。生物信息学软件预测分析发现TGEV感染宿主细胞时N蛋白主要定位于细胞核和线粒体，S（A-D）蛋白主要定位于细胞质和线粒体，S（A-D）蛋白具有7个优势B细胞抗原表位，N蛋白具有8个优势B细胞抗原表位。重组载体p-S（A-D）-His和p-N-His均在PK-15细胞内成功表达，且S（A-D）-His和N-His在PK-15细胞核和细胞质中均有分布。重组疫苗载体免疫小鼠后，免疫效果由高至低依次为p-N-His＞p-S（A-D）-His + p-N-His＞p-S（A-D）-His。结论本研究构建了TGEV的 S、N基因的DNA疫苗载体，免疫小鼠后均产生了较强的特异性抗体，为TGEV的核酸疫苗的研制提供了基础材料和依据。
- 猪传染性胃肠炎病毒 /
- S基因、 N基因 /
- 载体构建 /
- 免疫原性
Abstract: Objective In order to provide a technical support and basic data for the prevention and control of porcine transmissible gastroenteritis and the DNA vaccine research, the DNA vaccine vector of S and N genes of porcine transmissible gastroenteritis virus was constructed and its immunogenicity was tested. Method The A site and D site from the S gene and N gene was amplified from transmissible gastroenteritis virus and the N gene (alone) and the A and D sites (fusion) were cloned into the vaccine vector pCDNA3.1-His-C.Bioinformatics software was used to predict and analyze the secondary structure composition, tertiary configuration, subcellular localization and dominant B cell epitope of S(A-D) protein and N protein. The recombinant vectors were transfected into PK-15 cells, and expression distribution of the N gene (alone) and the A and D sites (fusion) was detected by indirect immunofluorescence and confocal detection. The recombinant vaccine vector was used to immunize mice alone or in combination, and the IgG antibody level was detected by indirect ELISA. Result The results showed that the A sites and D sites of S gene and N genes were amplified, and their sizes were 498bp, 606bp and 1149bp, respectively. the nucleic acid vaccine expression vectors p-S(A-D)-His and p-N-His for A and D sites (fusion) and N gene (alone) were constructed. Bioinformatics software predicted that when TGEV infected host cells, N protein was mainly located in the nucleus and mitochondria, S(A-D) protein was mainly located in the cytoplasm and mitochondria, S(A-D) protein had 7 dominant B cell epitopes, and N protein had 8 dominant B cell epitopes. Recombinant vectors p-S(A-D)-His and p-N-His were successfully expressed in PK-15 cells, and S(A-D)-His and N-His were distributed in the nucleus and cytoplasm of PK-15 cells. After immunizing mice with vaccine vector, the immune effect was p-N-His ＞ p-S(A-D)-His + p-N-His ＞ p-S(A-D)-His from high to low. Conclusion In this paper, the DNA vaccine vectors of S and N genes of TGEV were constructed, and strong specific antibodies were generated after immunizing mice, which provided the basic material and basis for the development of nucleic acid vaccine of TGEV.
- TGEV /
- S gene、N gene /
- vector construction /
- immunogenicity

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图 1 质粒p-S(A-D)-His构建模式

A：质粒p-S(A-D)-His图谱；B：A位点与D位点连接示意图；C：A位点与D位点连接碱基序列图。

Figure 1. The model diagram of plasmid p-S(A-D)-His construction

A: the plasmid p-S(A-D)-His profile; B: the diagram of the connection between site A and site D; C: the sequence diagram of connecting bases at sites A and D.

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图 2 TGEV S基因A、D位点扩增结果

A：A位点扩增结果；M为DNA分子质量标准DL2000，1为扩增的A位点。B：D位点扩增结果；M为DNA分子质量标准DL2000，1为扩增的D位点。

Figure 2. Amplification results of S gene A and D site of TGEV

A: amplification results of A site; M was DNA Marker DL2000, 1 was the PCR-amplified A site. B: amplification results of D site; M was DNA Marker DL2000, 1 was the PCR-amplified D site.

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图 3 TGEV N基因扩增结果

M：DNA分子质量标准DL2000；1、2：扩增的N基因。

Figure 3. The amplification results of TGEV N gene

M: DNA Marker DL2000; 1, 2: PCR-amplified N gene.

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图 4 S基因A位点重组载体p-S(A)-His的构建与鉴定

M：DNA分子质量标准DL2000；1、2：重组载体p-S(A)-His的单酶切；3、4：重组载体p-S(A)-His的双酶切。

Figure 4. Construction and identification of recombinant vectors p-S(A)-His at A site of S gene

M: DNA Marker DL2000; 1、2: single enzyme digestion of recombinant vector p-S(A)-His; 3、4: double digestion of recombinant vector p-S(A)-His.

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图 5 重组载体p-S（A-D）-His的构建与鉴定

M：DNA分子质量标准DL2000； 1：重组载体p-S(A-D)-His的单酶切；2：重组载体p-S(A-D)-His的双酶切。

Figure 5. Construction and identification of recombinant vectors p-S(A-D)-His

M：DNA Marker DL2000; 1:Single enzyme digestion of recombinant vector p-S(A-D)-His; 2: Double digestion of recombinant vector p-S(A-D)-His.

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图 6 重组载体p-N-His的构建与鉴定

M：DNA分子质量标准DL5000；1：重组载体p-N-His的双酶切。

Figure 6. Construction and identification of recombinant vectorsp-N-His

M: DNA Marker DL5000; 1: double digestion of recombinant vector p-N-His.

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图 7 S（A-D）蛋白、N蛋白三级结构同源建模

A：N蛋白结构图；B：N蛋白表面构像图；C：S（A-D）蛋白结构图；D：S（A-D）蛋白表面构像图。

Figure 7. The Homologous modeling of S(A-D)and N protein tertiary structure

A: structure chart of N protein; B: structure mapping of N protein; C: structure chart of S(A-D) protein; D: structure mapping of S(A-D) protein.

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图 8 N基因、S（A-D）片段表达的间接免疫荧光试验

Figure 8. The expression of N gene and S(A-D) fragments verified by indirect immunofluorescence assay

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表 1 A位点、D位点和N基因扩增引物序列

Table 1. The sequence of primers for amplification of A, D and N genes

基因 Gene	引物 Primers	序列（5′−3′） Sequence（5′−3′）	酶切位点 Restriction enzyme cutting site
S基因A位点 A-site in S gene	P1	CGCGGATCCATGTTAGTTACCAAACAGCCGT	Bam H I
S基因A位点 A-site in S gene	P2	CCGGAATTCTATTGTCCAGAAAACGTCAC	Eco R I
S基因D位点 D-site in S gene	P3	CCGGAATTCAAGTTGAAAACACAGCTATT	Eco R I
S基因D位点 D-site in S gene	P4	TGCTCTAGA ACTATTATCAGACGGTACACC	Xba I
N基因 N Gene	P5	CGCGGATCCATGGCCAACCAGGGAC	Bam H I
N基因 N Gene	P6	CCGGAATTCGTTCGTTACCTCATCAATT	Eco R I
表中加下划线的碱基序列为酶切位点序列。 The base sequences underlined in the table are the sequences of cleavage sites.

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表 2 小鼠分组及免疫程序

Table 2. Immunological grouping and immunological program of mice

组别 Group	疫苗载体种类 Vaccine carrier type	免疫剂量 Immunizing dose/μg	小鼠数量 Number of mice	免疫时间 Immune frequency	免疫位置 Immune site
1	p-S(A-D)-His	240	6	第1、7、21天	脚底板
2	p-N-His	240	6	第1、7、21天	脚底板
3	p-S(A-D)-His + p-N-His	120+120	6	第1、7、21天	脚底板
4	pCDNA3.1-His-C	240	6	第1、7、21天	脚底板

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表 3 S（A-D）蛋白和N蛋白亚细胞定位预测

Table 3. Prediction of subcellular localization of S(A-D) protein and N protein

组别 Group	亚细胞定位 Subcellular localization	可能性 Possibility/%
S（A-D）蛋白 S(A-D) protein	细胞质 Cytoplasm	34.8
	线粒体 Mitochondria	17.4
	细胞核 Cell nucleus	13.0
	质膜 Plasmalemma	13.0
	内质网 Endoplasmic reticulum	8.7
N蛋白 N protein	细胞核 Cell nucleus	65.2
	线粒体 Mitochondria	17.4
	细胞质 Cytoplasm	13.0
	溶酶体 Lysosome	4.3

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表 4 S（A-D）蛋白和N蛋白的B细胞抗原表位预测

Table 4. B cell epitope prediction of S(A-D) and N protein

蛋白 Protein	序号 Number	起始位点 Start site	结束位点 End site	序列 Amino acid sequence
S（A-D）蛋白 S(A-D)protein	1	33	43	FDQCNGAVLNN
	2	55	62	TTNVQSGK
	3	86	101	DSSFFSYGEIPFGVTD
	4	195	211	NLNNGFYPVSSSEVGLV
	5	233	250	LGMKRSGYGQPIASTLSN
	6	281	295	ALWDNIFKRNCTDVL
	7	306	318	CPFSFDKLNNYLT
N蛋白 N protein	1	4	32	QGQRVSWGDESTKTRGRSNSRGRKSNNIP
	2	43	87	QGSKFWNLCPRDFVPNGIGNRDQQIGYWNRQTRYRMVKGQRKELP
	3	101	108	ADAKFKDK
	4	118	146	DGAMNKPTTLGSRGANNESKALKFDGKVP
	5	150	189	QLEVNQSRDNSRSRSQSRSRSRNRSQSRGRQQSNNKKDDS
	6	201	244	LGVDTEKQQQRSRSKSKERSNSKTRDTTPKNENKHTWKRTAGKG
	7	251	271	GARSSSANFGDSDLVANGSSA
	8	316	378	DPKTEQFLQQINAYARPSEVAKEQRKRKSRSKSAERSEQEVVPDALIENYTDVFDDTQVEIID

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表 5 免疫小鼠抗TGEV血清IgG间接ELISA检测

Table 5. Indirect ELISA of anti-TGEV serum IgG in immunized mice

组别 Group	疫苗载体种类 Vaccine carrier type	免疫后不同时间抗TGEV血清IgG水平（OD_{450 nm}） Anti-tgev serum IgG levels at different time after immunization (OD_{450 nm})
组别 Group	疫苗载体种类 Vaccine carrier type	0天 0 days	14天 14 days	28天 28 days	42天 42 days
1	p-S（A-D）-His	0.133±0.011 a	0.218±0.0075 b	0.243±0.0060 c	0.401±0.0100 c
2	p-N-His	0.138±0.016 a	0.244±0.0025 a	0.344±0.0070 a	0.504±0.0141 a
3	p-S（A-D）-His + p-N-His	0.142±0.0050 a	0.225±0.0021 b	0.300±0.0050 b	0.471±0.0075 b
4	pCDNA3.1-His-C	0.134±0.0089 a	0.144±0.0076 c	0.141±0.0069 c	0.139±0.0020 d
同列数据肩标小写字母完全不同表示差异显著(P＜0.05)，含相同小写字母或无肩标表示差异不显著(P＞0.05) 。 Different lowercase letters of shoulder labels in the same column indicated significant differences (P＜0.05), while the same lowercase letters or no shoulder labels indicated no significant differences (P＞0.05).

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