传染性支气管炎病毒S1蛋白中和表位-Fc融合蛋白的表达及免疫原性鉴定

doi:10.11843/j.issn.0366-6964.2025.11.033

摘要/Abstract

摘要：

为构建禽传染性支气管炎病毒(IBV)S1蛋白的免疫原，本研究通过比对已公布的S1中和表位和当前我国流行的GI-19型代表毒株ck/CH/LDL/091022(LDL091022)S1蛋白氨基酸序列，鉴定第22—100和410—492位氨基酸区域为中和表位优势区，分别命名为B1和B2。构建表达LDL091022 B1和B2的质粒以及在C端融合鸡IgY Fc片段的质粒，通过Western blot和IFA检测发现融合IgY Fc片段后能够显著提高蛋白的表达量。转染Expi 293F细胞表达并纯化获得了融合IgY Fc片段的重组蛋白B1-Fc和B2-Fc。通过Western blot和ELISA鉴定B1-Fc和B2-Fc与LDL091022血清的反应性，结果发现B2-Fc与血清的反应性明显优于B1-Fc。将质粒pCAGGS-B2-Fc和重组蛋白B2-Fc免疫SPF鸡，通过ELISA和IFA检测血清中的抗体水平，结果显示B2-Fc加强免疫后能够有效诱导针对B2表位的抗体，且IFA结果显示血清能与LDL091022-S1蛋白和B2-Fc发生特异性反应。进一步利用中和试验检测血清中和效价为16。上述结果表明，本研究制备的IBV S1蛋白中和表位-Fc融合蛋白具有良好的表达水平和免疫原性，能够诱导机体产生一定水平的中和抗体，为进一步研发新型IBV亚单位疫苗提供了借鉴。

关键词: 禽传染性支气管炎病毒, S1亚基, 中和表位, IgY Fc片段, 免疫原性

Abstract:

To construct the immunogen of the S1 protein of avian infectious bronchitis virus (IBV), the amino acid sequences of the published S1 neutralizing epitopes were compared with the S1 protein of ck/CH/LDL/091022 (LDL091022), current prevalent GI-19 representative strain in China. The amino acid sequences at positions 22-100 and 410-492 of the S1 protein were recognized as principal neutralizing epitope areas, named as B1 and B2, respectively. Plasmids encoding LDL091022 B1 and B2 epitope peptides and plasmids with chicken IgY Fc fragment fused at the C-terminus of B1 and B2 were constructed. The significant increase of protein expression caused by IgY Fc fragment was demonstrated by indirect immunofluorescence assay (IFA) and Western blot assay. The recombinant plasmids were transfected into Expi 293F cells, and the recombinant proteins B1-Fc and B2-Fc with IgY Fc fragments were purified. Western blot and ELISA were used to assess the reactivity of B1-Fc and B2-Fc with LDL091022 serum, the results showed that B2-Fc had significantly better reactivity than B1-Fc. SPF chickens were immunized with the plasmid pCAGGS-B2-Fc and the recombinant protein B2-Fc. The serum antibody levels were detected by ELISA and IFA, the results demonstrated that B2-Fc booster immunization effectively induced antibodies against the B2 epitope. And the IFA result revealed the serum could react with LDL091022-S1 protein and B2-Fc. Moreover, the neutralization test showed that the serum neutralization titer was 16. These results demonstrated that the IBV S1 protein neutralizing epitope-Fc fusion protein prepared in this study showed good expression level and immunogenicity. It could induce certain levels of neutralizing antibodies, providing insights for the development of novel IBV subunit vaccines.

Key words: avian infectious bronchitis virus, S1 subunit, neutralizing epitope, IgY Fc fragment, immunogenicity

中图分类号:

S852.659.6

郭森, 李慧昕, 韩宗玺, 孙军峰, 夏长友. 传染性支气管炎病毒S1蛋白中和表位-Fc融合蛋白的表达及免疫原性鉴定[J]. 畜牧兽医学报, 2025, 56(11): 5758-5769.

GUO Sen, LI Huixin, HAN Zongxi, SUN Junfeng, XIA Changyou. Expression and Immunogenicity Analysis of the Fusion Protein Composed of IBV S1 Protein Neutralizing Epitope and Avian IgY Fc Fragment[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5758-5769.

图/表 12

表 1

已报道的IBV S1蛋白的中和表位"

毒株(登录号) Virus (Accession Number)	中和表位序列 Neutralizing epitope sequences	S1蛋白中位置 Position in S1 protein	参考文献 References
D207 (CAA41067.1)	NTYVYYYQSALRPPNGWHLQGGAYAVVNSTNYTN NAGSAEH CTVGV	24—61	^[15]
	IRISAMKNGTLFYNLTVS FHLYQTQTAQSGYYNFNLSFLSQFVYKASDFMYGSYH	132—149
	PSCSFRPETINSGLWFNSLSVSLAYGPLQGGCKQSVFSGR ATCCYAYSYNGPIACKGVYAGELQTNFECGL	291—398
	VTSCPYVSYGRFCIEPDGSLKM	546—567
Australian T strain (AY775779.1)	NYVYYYQSAFRPSGGWHLHGGAYAVVNVSQETSNAGS	25—61	^{[16, 17]}
	RIAAMKQGGNGPSDLFY	134—150
	QTYQTQTAQSGYYNFNFSFLSGFVYKEFNFMYGSYHPK CNFRPENINNGLWFNSLSVSLAYGPLQGGCKQSVFHGRATCC YAYSYLGPRLCKGVYSGELTQQFECG	298—404
SAIBk (DQ288927)	DNYVYYYQSAFRPPNGWHLQGGAYAVVNSINKTNNAGAASECS VGVPFNYTNGNDVGYNNSASSVAMTAPLPGMSWSRTQFCTAH CNFSDFTVFVTHCFANSCPLTGRIEANHIRISAMRNGSLFYN	24—150	^[18]
	QGGVQSILTYQTQTAQSGYYNFNLSFLSSFVYKESDYMYGSYHP ACNFRLETINNGLWFNSLSVSLAYGPLQGGCKQSVFSSRATCCYA YSYNGPRACKGVYAGELRQNFE	290—400
	STSENVTSCPYVSYGRFCIQPDGSIKQIVPQELENFVAPL LNVTENVLIPNSFNLTVTDEYIQTR	544—608
M41 (DQ834384.1)	SVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKVMREVKA LAYFVNGTAQDVILCDGSPRGLLACQYNTGNFSDGFYPFIN	166—247	^[19]
	SGGKLVGILTSRNET	501—515
	NCPYVSYGKFCIKPDGSIATIVP	545—567
	YTSNETTDVTS	175—185	^[20]
	IQTATEP	409—415	^[21]
	TAPSSGMALSSSQFCT	84—99
	DSSSYVYYYQSAFRPPNGWHLHGGAYAVVN	22—51
VicS (U29519.1)	QYNTGNFSDGLYPFTN	245—260	^[22]
	NCPYVSYGKFCIKPDGSIST	566—584
	SGGKLVGILTSRNET	518—532
	GSQAIENQFYIKLTNGS	532—537
QX(L1138) (KY933090.1)	YVYYYQSAFRPPNGWHLQGGAYAVVNSTN	25—54	^[23]
QX(L1138) (KY933090.1)	TVGVIKDVYNQSVASI	67—82
AY837465	AMTVPPAGMSWSVS	83—96
M41-CK(Mass) (MK728875.1)	VNQQFVVSGGKL	494—505
IBV-Gray (L14069.1)	GYNYGNFSDGFYPFTN	240—255	^[24]
QX(Sczy3) (JF732903)	PPQGMAW	87—93	^[25]
QX(Sczy3) (JF732903)	IQTRTEP	412—418	^[25]
CR88121 (JN542567.1)	NETNNAGSVSDCTAGT	54—69	^[20]
	VSNASPNSGGVD	279—290
	HPKCNFRPENI	328—338
YC181031	IQTRTEP	416—422	^[21]
	LDTSGSIDIFVVQGEYGLTYYKVNPCEDVNQQFVVSGGKLVGIL	465—509
	QFVVSGGNIVGMLTSRNETGSEQVENQFYVKLTNSSHRRKRSID QNVTSCSYVSYGRFCIEPDGSLKK	504—571

表 1

表 2

图 1

图 2

图 3

图 4

图 5

图 6

图 7

图 8

图 9

图 10

参考文献 30

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引物 Primers	引物序列(5′→3′) Primers sequences
B1-F	AAGAATTGTGCGGCCGCATGTTGGGGAAGTCACTGTTTTTAGTGACCATTTTGTGTGCACTATGTAGTGCAgattctgccaataattatgtgtactactac
B2-F	AAGAATTGTGCGGCCGCATGTTGGGGAAGTCACTGTTTTTAGTGACCATTTTGTGTGCACTATGTAGTGCAtctcgtatacagactagaacgg
B1-R1	CCATGGTGGCCTCGAGCTA$\underline{{\rm{GTGGTGATGGTGATGGTG}}}$actacaaaactgtgacttagacc
B2-R1	CCATGGTGGCCTCGAGCTA$\underline{{\rm{GTGGTGATGGTGATGGTG}}}$gctattggttaacttaacataaaactgg
B1-R2	GGAGCCACCGCCACCAGAGCCACCACCGCCactacaaaactgtgacttagaccaag
B2-R2	GGAGCCACCGCCACCAGAGCCACCACCGCCgctattggttaacttaacataaaactgg
CHINy-F	CTGGTGGCGGTGGCTCCGGCGGTGGTGGCTCTGCCTCTCCTACCAGCC
CHINy-R	CCATGGTGGCCTCGAGTTACTA$\underline{{\rm{GTGGTGATGGTGATGGTG}}}$CTTGCCTGCCTGCTTC

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