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

doi:10.7668/hbnxb.20193768

Abstract

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

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.

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Figures/Tables 13

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

Fig.1 The prediction of transmembrane region of NS3 protein

Fig.2 The prediction of signal peptide of NS3 protein

Fig.3 The prediction of tertiary structure of NS3 protein

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

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

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

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.

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.

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

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

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.

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

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