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

Vector Construction and Immunogenicity of S and N Gene DNA Vaccine 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 DNA vaccine vector of S and N genes of porcine transmissible gastroenteritis virus (TGEV) was constructed with the vaccine immunogenicity detemined to pave the way for studying, preventing, and controling TGE. Method A and D sites on S and N from a TGEV were amplified. The N gene alone as well as 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, tertiary configuration, subcellular localization, and dominant B cell epitope of S (A-D) and N proteins. The recombinant vectors were transfected into PK-15 cells, and expression distribution of N and the A and D sites fusion detected by indirect immunofluorescence and confocal detection. Mice were immunized with the single or combined recombinant vaccine vector to detect the IgG antibody using indirect ELISA. Result The A and D sites of the S were 498bp and 606bp, respectively, and the N, 1,149bp in length. The nucleic acid vaccine expression vectors p-S (A-D)-His and p-N-His for the A and D sites (fusion) and N were constructed. Bioinformatics software predicted that, when TGEV infected the host cells, N protein was mainly located in the nucleus and mitochondria and S (A-D) largely in the cytoplasm and mitochondria, while S (A-D) had 7 and N, 8 dominant B cell epitopes. All p-S (A-D)-His and p-N-His were successfully expressed in PK-15 cells distributed in the nucleus and cytoplasm. The immunized mice showed an effect of immunity in the order of p-N-His＞p-S (A-D)-His + p-N-His＞p-S (A-D)-His. Conclusion The DNA vaccine vectors of S and N of TGEV were successfully constructed. Strong specific antibodies were generated in lab mice after the immunization.
- TGEV /
- S and 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. Amplified A and D sites of TGEV S gene

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. Amplified N gene of TGEV

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 p-S (A)-His and p-S (D)-His at A and D sites of S gene

M1: DNA marker DL2000; M2: DNA marker DL10000; 1: single enzyme digestion of recombinant vector p-S (A)-His; 2: single enzyme digestion of recombinant vector p-S (D)-His; 3 and 4: double digestion of recombinant vector p-S (A)-His; 5 and 6: double digestion of recombinant vector p-S (D)-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 vectors p-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. Homologous modeling on tertiary structures of S (A-D) and N protein

A: Structure of N protein; B: image of N protein structure; C: image of S (A-D) protein structure; D: image of S (A-D) protein structure.

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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. Expressions of N and S (A-D) fragments verified by indirect immunofluorescence assay

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表 1 S基因A位点、D位点和N基因扩增引物序列

Table 1. Sequences of primers for amplifications of A and D sites and N gene

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。 Cleavage sites are underlined.

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表 2 小鼠分组及免疫程序

Table 2. Groups and procedures of mice immunization

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. Predicted subcellular localization of S (A-D) and N proteins

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. Predicted B cell epitope of S (A-D) and N proteins

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. Anti-TGEV serum IgG in immunized mice detected by indirect ELISA

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）。 Data with different lowercase letters on same column indicate significant differences at P＜0.05; those with or without same lowercase letters indicate no significant differences at P＞0.05.

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