牛病毒性腹泻病毒<i>NS3</i>基因的生物信息学分析、蛋白纯化及免疫原性分析

doi:10.7668/hbnxb.20193768

摘要/Abstract

摘要：

为了探讨牛病毒性腹泻病毒(BVDV)非结构蛋白NS3(P80),并获得具有较高免疫原性的NS3重组蛋白,利用生物信息学研究方法对NS3蛋白氨基酸进行序列分析;经PCR技术扩增NS3基因序列;通过无缝克隆技术构建pET-22b(+)-NS3重组表达质粒,将其转化至大肠杆菌BL21(DE3)感受态细胞中,诱导其表达 NS3 重组蛋白并提纯;通过Western Blotting方法检测其反应原性;将获得的较高纯度的NS3重组蛋白免疫BABL/c小鼠,收集血清,ELISA方法分别检测血清中IgG、IgG1和IgG2a抗体水平;最后通过病毒中和试验检测其中和病毒的能力。结果表明:NS3蛋白氨基酸序列中无信号肽和跨膜区,二级结构主要以无规则卷曲为主,且NS3氨基酸序列中含有优势抗原表位;利用PCR和无缝克隆技术成功构建了pET-22b(+)-NS3重组表达载体,经诱导表达获得了较高纯度的NS3重组蛋白,大小约为75 ku,与理论大小相符;Western Blotting结果表明,NS3重组蛋白具有较高的反应原性;ELISA检测NS3重组蛋白免疫后的小鼠能够产生高水平的IgG、IgG1和IgG2a抗体,结果显示,免疫组小鼠产生的抗体水平极显著高于PBS阴性对照组(P<0.01);血清中和抗体水平也极显著高于PBS阴性对照组(P<0.01)。总之,成功获得了纯度高且具有免疫原性的NS3重组蛋白。

关键词: 牛病毒性腹泻病毒, NS3, 生物信息学, 原核表达, 免疫原性

Abstract:

To explore the non-structural protein NS3(P80)of Bovine viral diarrhea virus (BVDV),and to obtain high immunogenic NS3 recombinant protein,the amino acid sequence of NS3 protein was analyzed by bioinformatics software.The sequence of NS3 gene was amplified by PCR,and the recombinant expression vector pET-22b(+)-NS3 was constructed by seamless cloning technology.The recombinant expression vector was transformed into competent cells of E.coli BL21(DE3) and induced to express NS3 recombinant protein.The reactivity of NS3 recombinant protein was detected by Western Blotting.BABL/c mice were immunized with the obtained high purity NS3 recombinant protein,and the serum was collected.The levels of IgG,IgG1 and IgG2a antibodies in serum were detected by ELISA,and the ability of neutralizing virus was detected by virus neutralization test.The results showed that there was no signal peptide and transmembrane region in the amino acid sequence of NS3 protein,the secondary structure was mainly random coil,and the NS3 amino acid sequence contained dominant antigen epitopes.The recombinant expression vector pET-22b(+)-NS3 was successfully constructed by PCR and seamless cloning techniques,and the recombinant protein of NS3 with high purity was obtained by inducing expression,with the size of about 75 ku,which was consistent with the theoretical size.Western Blotting results showed that the NS3 recombinant protein had high reactivity.ELISA test showed that mice immunized with NS3 recombinant protein could produce high levels of IgG,IgG1 and IgG2a antibodies.The antibody levels of mice immunized with NS3 recombinant protein were extremely significantly higher than those of PBS negative control group(P<0.01),and neutralizing antibody level was also significantly higher than that of PBS negative control group(P<0.01).In conclusion,this study successfully obtained NS3 recombinant protein with high purity and immunogenicity.

Key words: Bovine viral diarrhea virus, NS3, Bioinformatics, Prokaryotic expression, Immunogenicity

杨宁宁, 徐明国, 张江伟, 易继海, 陈创夫. 牛病毒性腹泻病毒NS3基因的生物信息学分析、蛋白纯化及免疫原性分析[J]. 华北农学报, 2023, 38(4): 205-212. doi: 10.7668/hbnxb.20193768.

YANG Ningning, XU Mingguo, ZHANG Jiangwei, YI Jihai, CHEN Chuangfu. Bioinformatics Analysis,Protein Purification and Immunogenicity Analysis of NS3 Gene of Bovine viral diarrhea virus[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(4): 205-212. doi: 10.7668/hbnxb.20193768.

http://www.hbnxb.net/CN/Y2023/V38/I4/205

图/表 13

表1 生物信息学分析软件

Tab.1 Bioinformatics analysis software

软件名称 Software name	网址 Website	预测目的 Prediction purpose
VaxiJen v2.0	http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html	保护性抗原预测
ProtParam	https://web.expasy.org/protparam/	蛋白理化特性
TMHMM Serverv.2.0	http://www.cbs.dtu.dk/services/TMHMM/	跨膜结构域
SignalP 4.1 server	http://www.cbs.dtu.dk/services/SignalP-4.1/	信号肽
SOPMA	https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl	二级结构
phyre2	http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index	三级结构
IEBD	http://www.iedb.org/	B细胞抗原表位

图1 NS3蛋白跨膜结构域预测

Fig.1 The prediction of transmembrane region of NS3 protein

图2 NS3蛋白信号肽预测

Fig.2 The prediction of signal peptide of NS3 protein

图3 NS3蛋白的二级结构预测

Fig.3 The prediction of tertiary structure of NS3 protein

图4 NS3蛋白的三级结构预测模型

Fig.4 The prediction model of tertiary structure of NS3 protein

表2 预测多肽

Tab.2 Predicted peptides

序号 No.	开始 Start	结束 End	肽 Peptide	长度 Length
1	7	16	KITEHEKCHV	10
2	23	33	TAFFGIMPRGT	11
3	46	49	LKVR	4
4	56	62	WAYTHQG	7
5	92	119	NNKLTDETEYGVKTDSGCPDGARCYVLN	28
6	128	215	SKGAVVHLQKTGGEFTCVTASGTPAFFDLKNLKGWSGLPIFEASSGRVVGRVKVGKNE	88
			ESKPTKIMSGIQTVSKSTADLTEM
7	229	232	GAGK	4
8	282	288	DMKEGDM	7
9	306	312	PKLRAAM	7
10	358	383	TTTGQKHPIEEFIAPEVMKGEDLGSQTTTGQKHPIEEFIAPEVMKGEDLGSQ	26
11	392	398	IPTEEMK	7
12	420	442	AKGYNSGYYYSGEDPASLRVVTS	23
13	473	488	CEKRVRVSSKIPFIVT	16
14	499	512	EQAQRRGRVGRVKP	14
15	522	537	ATGSKDYHYDLLQAQR	16
16	551	565	REMNYDWSLYEEDSL	15
17	567	567	I	1
18	594	640	DHPEPIQLAYNSYEVQVPVLFPKIRNGEVTDTYENYSFLNARKLGED	47
19	650	652	DED	3
20	662	672	WPDPGNQQVVE	11

图5 NS3基因的PCR扩增 M.DM10000 Marker;1.阴性对照;2—3.NS3目的基因。

Fig.5 Amplification of NS3 gene by PCR M.DM10000 Marker;1.Negative control;2—3.NS3 target gene.

图6 pET-22b(+)-NS3重组质粒双酶切鉴定 M.1 kb DNA Marker;1.pET-22b(+)质粒;2.pET-22b(+)-NS3重组质粒双酶切。

Fig.6 The double enzyme identification of pET-22b(+)-NS3 recombinant plasmid M.1 kb DNA Marker;1.pET-22b(+)plasmid control;2.pET-22b (+)-NS3 recombinant plasmid double enzyme digestion.

图7 NS3重组蛋白最佳诱导时间及可溶性分析 A:M.蛋白质分子质量标准;1—5.pET-22b(+)-NS3 37 ℃ 诱导0,2,4,6,8 h的菌液。B:M.蛋白质分子质量标准;1.破碎后菌液沉淀;2.破碎后菌液上清液。

Fig.7 Optimum induction time and compatibility analysis of recombinant protein NS3 A:M.Protein molecular weight standard;1—5.pET-22b(+)-NS3 37 ℃ induces 0,2,4,6,8 h bacterial liquid.B:M.Protein molecular weight standard;1.Precipitation of bacterial liquid after crushing;2.Supernatant of bacterial liquid after crushing.

图8 NS3重组蛋白的纯化鉴定 M.蛋白质分子质量标准;1.纯化后的NS3重组蛋白。

Fig.8 Purification and identification of NS3 recombinant protein M.Protein molecular weight standard;1.Purified NS3 recombinant protein.

图9 NS3重组蛋白的Western Blotting鉴定 M.蛋白质分子质量标准;1—2.纯化后的NS3重组蛋白。

Fig.9 Western Blotting identification of NS3 recombinant protein M.Protein molecular weight standard;1—2.Purified NS3 recombinant protein.

图10 小鼠血清IgG、IgG1和IgG2a抗体检测 A.小鼠血清中IgG抗体水平;B.小鼠血清中IgG1抗体水平;C.小鼠血清中IgG2a抗体水平。不同大写字母表示1%水平差异极显著(P<0.01)。图11同。

Fig.10 Detection of IgG,IgG1 and IgG2a antibodies in mice serum A.The level of IgG antibody in mouse serum;B.The level of IgG1 antibody in mouse serum;C.The level of IgG2a antibody in mouse serum. The different capital letters are extremely significantly different at the 1% level(P<0.01).The same as Fig.11.

图11 免疫小鼠血清中病毒中和抗体检测

Fig.11 Detection of viral neutralizing antibodies in the serum of immunized mice

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牛病毒性腹泻病毒NS3基因的生物信息学分析、蛋白纯化及免疫原性分析

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牛病毒性腹泻病毒NS3基因的生物信息学分析、蛋白纯化及免疫原性分析

Bioinformatics Analysis,Protein Purification and Immunogenicity Analysis of NS3 Gene of Bovine viral diarrhea virus

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