鲤疱疹病毒3型ORF57基因工程亚单位疫苗制备及免疫保护研究

doi:10.3969/j.issn.1004-1524.2022.10.08

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (10): 2138-2148.DOI: 10.3969/j.issn.1004-1524.2022.10.08

鲤疱疹病毒3型ORF57基因工程亚单位疫苗制备及免疫保护研究

刘舒亚¹(), 何汶璐², 陶雨¹, 黄双慧¹, 任永强¹, 耿毅¹, 黄小丽³, 陈德芳³, 欧阳萍¹^,^*()

1.四川农业大学动物医学院,四川成都 611130
2.西南医科大学附属中医医院中葡中医药国际合作中心,四川泸州 646099
3.四川农业大学动物科技学院,四川成都 611130

收稿日期:2021-04-20 出版日期:2022-10-25 发布日期:2022-10-26
通讯作者: 欧阳萍
作者简介:*欧阳萍,E-mail: ouyang.ping@live.cn
刘舒亚(1997—),女,四川成都人,硕士研究生,主要从事水生动物疾病研究。E-mail: liushuya0927@163.com
基金资助:
四川省国际科技创新合作项目(2020YFH0156)

Preparation and immunoprotection of ORF57 subunit vaccine of Cyprinid herpesvirus 3

LIU Shuya¹(), HE Wenlu², TAO Yu¹, HUANG Shuanghui¹, REN Yongqiang¹, GENG Yi¹, HUANG Xiaoli³, CHEN Defang³, OUYANG Ping¹^,^*()

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. China Portugal International Cooperation Center of Traditional Chinese Medicine, Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University, Luzhou 646099, Sichuan, China
3. College of Animal Science, Sichuan Agricultural University, Chengdu 611130, China

Received:2021-04-20 Online:2022-10-25 Published:2022-10-26
Contact: OUYANG Ping

摘要/Abstract

摘要：

旨在研究鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3)囊膜蛋白ORF57作为疫苗候选抗原的可能性。根据已经发表的鲤疱疹病毒3型全基因组(GenBank登录号为 DQ657948.1),设计特异性引物,扩增、克隆ORF57序列并进行生物信息学分析。构建重组原核表达载体pET-32a(+)-CyHV-3-ORF57,大肠埃希菌体外表达获得重组蛋白ORF57。此外,对ORF57亚单位疫苗的免疫保护性进行研究。鲤鱼免疫4周后CyHV-3攻毒具有75%的存活率,ORF57亚单位疫苗组鲤鱼的存活率显著高于PBS和pET-32a(+)组。实验结果证明,重组ORF57亚单位疫苗具有较强的免疫保护性,为免疫学临床的应用和CyHV-3疫苗的开发提供了实验基础。

关键词: 鲤疱疹病毒3型, 重组蛋白, 亚单位疫苗, 免疫保护

Abstract:

The aim of this study was to study the possibility of ORF57, an important capsule protein of Cyprinid herpesvirus 3, as a vaccine candidate antigen. According to the published genome sequence of Cyprinid herpesvirus 3 (GenBank number: DQ657948.1), specific primers were designed to amplify and clone the ORF57 sequence, and bioinformatics analysis was performed. The recombinant prokaryotic expression vector pET-32a(+) -CyHV-3-ORF57 was constructed, and the recombinant protein ORF57 was expressed by E. coli in vitro. In addition, the immune protection of ORF57 subunit vaccine was studied. The survival rate of CyHV-3 was 75% after 4 weeks of immunization, and the survival rate of ORF57 subunit vaccine group was significantly higher than that of PBS and PET-32a (+) groups. The results showed that the recombinant ORF57 subunit vaccine had strong immune protection, which provided an experimental basis for the clinical application of immunology and the development of CyHV-3 vaccine.

Key words: Cyprinid herpesvirus 3, recombinant protein, subunit vaccine, immune protection

中图分类号:

S941.41

刘舒亚, 何汶璐, 陶雨, 黄双慧, 任永强, 耿毅, 黄小丽, 陈德芳, 欧阳萍. 鲤疱疹病毒3型ORF57基因工程亚单位疫苗制备及免疫保护研究[J]. 浙江农业学报, 2022, 34(10): 2138-2148.

LIU Shuya, HE Wenlu, TAO Yu, HUANG Shuanghui, REN Yongqiang, GENG Yi, HUANG Xiaoli, CHEN Defang, OUYANG Ping. Preparation and immunoprotection of ORF57 subunit vaccine of Cyprinid herpesvirus 3[J]. Acta Agriculturae Zhejiangensis, 2022, 34(10): 2138-2148.

图/表 10

表1 CyHV-3 ORF57序列的特异性引物

Table 1 Specific primers for CyHV-3 ORF57

引物名称 The name of the primer	引物序列 Primer sequence (5'-3')	酶切位点 Restriction enzyme cutting site
F1	GGATCCATGGCAGCCAGGGA	BamH Ⅰ
R1	AAGCTTGGATTTGGTTTTGGGTAGGTTC	Hind Ⅲ

图1 ORF57氨基酸序列的组成分析、亲/疏水性分析以及跨膜区和信号肽的预测 A,ORF57氨基酸序列的组成分析;B,ORF57氨基酸序列的亲/疏水性分析;C,TMHMM程序对ORF57氨基酸序列的跨膜区预测;D,信号肽预测。

Fig.1 ORF57 amino acid sequence composition analysis, affinity/hydrophobicity analysis and prediction of transmembrane domain and signal peptide A, Composition analysis of ORF57 amino acid sequence; B, Affinity/hydrophobicity analysis of ORF57 amino acid sequence; C, Prediction of transmembrane region of ORF57 amino acid sequence by TMHMM; D, Prediction of signal peptide.

图2 CyHV-3ORF57氨基酸序列的磷酸化位点预测

Fig.2 Prediction of phosphorylation site of CyHV-3ORF57 amino acid sequence

图3 CyHV-3 ORF57蛋白的二级结构(A)和三级结构(B)预测 h,α-螺旋;e,β-折叠;t,β-转角;c,无规则卷曲。

Fig.3 Secondary structure (A) and tertiary structure (B) of CyHV-3 ORF57 protein h, α-helix; e, β-sheet; t, β-turn; c, Random coil.

图4 CyHV-3 ORF57序列的PCR扩增和T载体重组克隆质粒鉴定 A图中:M,DNA Marker 10000;1,ORF57序列扩增产物大小为1 449 bp;2,阴性对照。B图中:M,DNA Marker 10000;1,pMD19-T-CyHV-3-ORF57双酶切鉴定;2,pMD19-T-CyHV-3-ORF57单酶切鉴定。

Fig.4 The PCR amplication result of the CyHV-3 ORF57 sequence and identification of cloing plasmid In Fig. A: M, DNA Marker 10000; 1, ORF57 amplification product; 2, Negative control. In Fig. B, M, DNA Marker 10000; 1, pMD19-T-CyHV-3-ORF57 double digestion identification; 2, pMD19-T-CyHV-3-ORF57 single digestion identification.

图5 CyHV-3-ORF57重组表达质粒的酶切鉴定1、2,pET-32a(+);3、4,pMD19-T-CyHV-3-ORF57双酶切。

Fig.5 Digestion identification of CyHV-3-ORF57 recombinant expression plasmid1, 2, pET-32a (+); 3, 4, pMD19-T-CyHV-3-ORF57 double digestion.

图6 重组蛋白ORF57的诱导表达 M,蛋白质Marker;1、2,IPTG诱导的BL21-pET32a菌液的上清;3、4,IPTG诱导的BL21-pET32a菌液的沉淀。

Fig.6 Expression of recombinant protein ORF57 M, Protein Marker; 1, 2, Induced supernatant of BL21-pET32a by IPTG; 3, 4, Induced precipitation of BL21-pET32a by IPTG.

图7 重组蛋白ORF57的纯化和Western-blot验证 A图中:M,蛋白质Marker;1,纯化所得目的蛋白条带。B图中:M,蛋白质Marker;1,目的蛋白免疫印迹。

Fig.7 Purification and Western-blot analysis of ORF57 In Fig. A: M, Protein Marker; 1, Purified protein. In Fig. B: M, Protein Marker; 1, Target protein.

图8 攻毒后的累计存活率

Fig.8 Cumulative survival rate after infection

图9 ELISA法检测鲤鱼血清抗体水平不同柱上没有相同小写字母表示差异显著(P<0.05)。

Fig.9 The carp serum antibody levels detected by ELISA The bars without the same lowercase letters indicated the significant difference(P<0.05).

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鲤疱疹病毒3型ORF57基因工程亚单位疫苗制备及免疫保护研究

Preparation and immunoprotection of ORF57 subunit vaccine of Cyprinid herpesvirus 3

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