诱导型表达H5N1亚型禽流感病毒HA蛋白的乳酸乳球菌的构建及其对鸭的免疫原性分析

doi:10.11843/j.issn.0366-6964.2025.02.027

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (2): 774-787.doi: 10.11843/j.issn.0366-6964.2025.02.027

诱导型表达H5N1亚型禽流感病毒HA蛋白的乳酸乳球菌的构建及其对鸭的免疫原性分析

吴佳辉(), 沈世彦(), 邓锦波, 吴海阳, 任芷欣, 吴杨博, 黄娟, 黄浩滨, 潘伟雄, 赵锃珏, 何容肖, 孙崇军, 张玲华*()

华南农业大学生命科学学院, 广东省农业生物发育与环境适应重点实验室, 广州 510642

收稿日期:2024-03-22 出版日期:2025-02-23 发布日期:2025-02-26
通讯作者: 张玲华 E-mail:1142620951@stu.scau.edu.cn;EnshWinter@163.com;lhzhang@scau.edu.cn
作者简介:吴佳辉(1999-)，男，广东普宁人，博士生，主要从事微生物与免疫研究，E-mail: 1142620951@stu.scau.edu.cn
沈世彦(2001-)，男，湖南娄底人，硕士生，主要从事微生物学和合成生物学研究，E-mail: EnshWinter@163.com
第一联系人：
吴佳辉和沈世彦为同等贡献作者
基金资助:
校企合作产学研项目(h2019364；h20220334)

Construction of Recombinant Lactococcus lactis Inducible Expressing HA Protein of H5N1 Subtype Avian Influenza Virus and Analysis of Its Immunogenicity in Ducks

WU Jiahui(), SHEN Shiyan(), DENG Jinbo, WU Haiyang, REN Zhixin, WU Yangbo, HUANG Juan, HUANG Haobin, PAN Weixiong, ZHAO Zengjue, HE Rongxiao, SUN Chongjun, ZHANG Linghua*()

Guangdong Provincial Key Laboratory for the Development Biology and Environmental Adaptation of Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong 510642, China

Received:2024-03-22 Online:2025-02-23 Published:2025-02-26
Contact: ZHANG Linghua E-mail:1142620951@stu.scau.edu.cn;EnshWinter@163.com;lhzhang@scau.edu.cn

摘要/Abstract

摘要：

禽流感是由禽流感病毒引起的高度传染性疾病，受到感染的雏禽死亡率高，给家禽健康和经济带来严重影响。尽管传统疫苗可以有效预防禽流感，但由于鸭类养殖方式多为散户散养，环境多变，在接种传统疫苗时存在操作繁琐和免疫应激强等问题。为了解决此难题，本研究旨在开发一种便捷且安全的雏鸭血凝素(hemagglutinin, HA)口服生物制剂。我们通过构建系列锚定序列(pgsA、BmpA、cA和M6)-GFP报告表达系统，可视化地评估了不同类型锚定序列对乳酸菌乳球表面展示系统的效果，成功筛选出BmpA和cA作为乳酸菌乳球中的高效锚定序列。比较优化密码子前后的HA在乳酸乳球菌的表达效果后，选取优化密码子的HA基因序列与BmpA、cA连接，构建乳酸菌表达质粒。然后把构建好的表达质粒转入基因组整合HA1基因的乳酸乳球菌中，从而得到诱导型分泌联合表面展示HA蛋白的重组乳酸乳球菌(NZB-HA1-pNZ8148-BmpA-HA1和NZB-HA1-pNZ8148-HA1-cA)。ELISA检测结果表明，与口服单一表达型的重组乳酸乳球菌相比，口服诱导型分泌-表面展示乳酸乳球菌能诱生雏鸭血清中更佳的HA特异性IgG水平。本研究成功构建了锚定序列-GFP乳酸菌筛选系统，利用其筛选出了适合乳酸乳球菌的高效锚定序列。并将HA蛋白通过分泌联合表面展示的方式在乳酸乳球菌进行复合表达，成功获得了对雏鸭具有良好HA免疫原性的口服生物制剂，为后续开发操作简便、安全的鸭禽流感口服疫苗提供可行的实践路径。

关键词: 锚定序列, 乳酸乳球菌, 禽流感, 雏鸭, HA

Abstract:

Avian influenza, caused by avian influenza viruses, is a highly contagious disease characterized by high mortality rates among infected poultry, posing significant health risks to poultry and economic burdens. However, the majority of duck farming comprises small-scale and free-range practices, where environmental conditions vary and ducks are prone to diseases. In such scenarios, traditional vaccination practices present challenges such as inconvenient injection and immunological stress. To tackle this issue, this study devised a method to produce a convenient and safe oral biological agent of hemagglutinin (HA) tailored specifically for ducklings. We constructed the anchoring sequence (pgsA, BmpA, cA, and M6)-GFP reporter expression system, enabling the visualization of different anchoring sequences' effects on surface-displayed proteins in Lactococcus lactis (L. lactis). With this system, we successfully identified BmpA and cA as efficient anchoring sequences in L. lactis. After comparing the expression efficiency of HA in L. lactis and optimizing the codons, we selected the optimized HA gene sequence and connected it with BmpA and cA. Then, we co-integrated them into the expression plasmids of L. lactis. Subsequently, we introduced the constructed expression plasmids into L. lactis containing the integrated HA1 gene in the genome, thereby generating recombinant L. lactis capable of both inducible secretory expression and surface display of HA(NZB-HA1-pNZ8148-BmpA-HA1 and NZB-HA1-pNZ8148-HA1-cA). The ELISA results indicated that orally administered inducible secretory-surface display recombinant L. lactis eliciteds superior levels of HA-specific IgG in duckling serum compared to single-expression recombinant L. lactis treatment. This study successfully established the Anchoring sequence-GFP screening system for Lactobacillus, identifying efficient anchoring sequences suitable for L. lactis, thereby achieving efficient expression of HA on the surface of L. lactis. Subsequently, in conjunction with the genomic secretion expression, we successfully obtained an oral biological agent with satisfactory HA immunogenicity for ducklings. This achievement offers a practical and feasible strategy for the subsequent development of convenient and safe avian influenza oral vaccines.

Key words: anchoring sequence, Lactococcus lactis, avian influenza, duckling, HA

中图分类号:

S834
Q939.94

吴佳辉, 沈世彦, 邓锦波, 吴海阳, 任芷欣, 吴杨博, 黄娟, 黄浩滨, 潘伟雄, 赵锃珏, 何容肖, 孙崇军, 张玲华. 诱导型表达H5N1亚型禽流感病毒HA蛋白的乳酸乳球菌的构建及其对鸭的免疫原性分析[J]. 畜牧兽医学报, 2025, 56(2): 774-787.

WU Jiahui, SHEN Shiyan, DENG Jinbo, WU Haiyang, REN Zhixin, WU Yangbo, HUANG Juan, HUANG Haobin, PAN Weixiong, ZHAO Zengjue, HE Rongxiao, SUN Chongjun, ZHANG Linghua. Construction of Recombinant Lactococcus lactis Inducible Expressing HA Protein of H5N1 Subtype Avian Influenza Virus and Analysis of Its Immunogenicity in Ducks[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 774-787.

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名称 Names	特征 Characteristics	来源 Reference or source
质粒Plasmids
pNZ8148	乳酸菌表达质粒	本实验室保存
pUC-pgsA	锚定序列	本实验室保存
pUC-BmpA	锚定序列	本实验室保存
pUC-GW-cA-M6	锚定序列	本实验室保存
pUC19-GFP		本实验室保存
pUC57-HA0	带有信号肽序列SPUsp45	苏州金唯智生物科技有限公司合成
pUC57-HA1	HA乳酸菌偏好性密码子优化	苏州金唯智生物科技有限公司合成
pJW4.1n	乳酸菌基因组敲入质粒	陆军军医大学王竞老师惠赠^[15]
菌株Strains
大肠杆菌DH5α		本实验室保存
大肠杆菌MC1061		本实验室保存
乳酸乳球菌NZB	LacZ基因整合到基因组	陆军军医大学王竞老师惠赠^[15]

名称	引物序列(5′→3′)
Name	Primer sequences
SP F	ATGAAAAAAAAGATTATCTCAGCTATTTT
SP R	CAATGCAAATTTGATCAGCGTAAACACCTGA
HA0 F	ATAAGGAGGCACTCAAAATGAAAAAAAAGATTATCTC
HA0 R	CATATTAAATATTCTCCTCTTAAATGCAAATTCTGCATT
HA1 F	TCAGGTGTTTACGCTGATCAAATTTGCATTGGCTA
HA1 R	CATATTAAATATTCTCCTCTAAATACAAATTCTACATTG
SP-HA-Xho I F	CCGCTCGAGCGGATGAAAAAAAAG
HA-Xba I R	GCTCTAGAGCTTAAATGCAAATTCTGC
Pz F	AAGTCGCGTGGCGCGCCAGTCTTATAACTATACTGACAATA
Ter R	GAAATGATAGGCGCGCCATAAGCAAAAGGCAGCTGAT
pgsA F	ATGAAAAAAGAACTGAGCTTTCATGAAAAGCTGC
pgsA R	TGAACCACCACCACCGGAT
GFP-CM F	ATGAAAAAAAAGATTATCTCAGCTATTTTAA
GFP-CM R	TTATTTTATTCGTAGATACTGACCAATTAAAATAGTATCGC
8148 F	CCTGCCCCGTTAGTTGAAGAAGG
8148 R	GCTTTATCAACTGCTGCTTTTTGGCT
BmpA-HA1(1)F	AAATAAATTATAAGGAGGCACTCACATGAAAAAACGCGTAATCGCAGTTAG
BmpA-HA1(1)R	AAATTTGATCTGAACCACCACCACCGGAT
BmpA-HA1(2)F	TGGTGGTTCAGATCAAATTTGCATTGGCTACCATGC
BmpA-HA1(2)R	GTTTTCTAATTTTGGTTCAAAGAATTAAATACAAATTCTACATTGTAATGATCCATTTG
HA1-cA(1)F	ATAAATTATAAGGAGGCACTCACATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCT
HA1-cA(1)R	CTCCTCCTCCAATACAAATTCTACATTGTAATGATCCATTTGAACACATC
HA1-cA(2)F	AATTTGTATTGGAGGAGGAGGATCCGGTGGT
HA1-cA(2)R	GGTTTTCTAATTTTGGTTCAAAGAATTATTTTATTCGTAGATACTGACCAATTAAAATA
SPHA1-8148 F	TAAATTATAAGGAGGCACTCACATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTA
SPHA1-8148 R	GTTTTCTAATTTTGGTTCAAAGAATTAAATACAAATTCTACATTGTAATGATCCATTTG
SP-GFP F	GTTTACGCTCGTAAAGGCGAAGAGCTGTTCAC
Nco I-pgsA F	AAATAAATTATAAGGAGGCACTCACATGAAAAAAGAACTGAGCTTTCATGAAAAGCT
Nco I-BmpA F	AAATAAATTATAAGGAGGCACTCACATGAAAAAACGCGTAATCGCAGTTAG

组别 Group	剂量 Dose	试验动物/只 Animal numbers
PBS组	100 μL·只^-1	5
NZB-pNZ8148-HA1免疫组	1.0×10¹¹ CFU·只^-1	5
NZB-pNZ8148-BmpA-HA1免疫组	1.0×10¹¹ CFU·只^-1	5
NZB-pNZ8148-HA1-cA免疫组	1.0×10¹¹ CFU·只^-1	5
NZB-HA1-pNZ8148-BmpA-HA1免疫组	1.0×10¹¹ CFU·只^-1	5
NZB-HA1-pNZ8148-HA1-cA免疫组	1.0×10¹¹ CFU·只^-1	5

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诱导型表达H5N1亚型禽流感病毒HA蛋白的乳酸乳球菌的构建及其对鸭的免疫原性分析

Construction of Recombinant Lactococcus lactis Inducible Expressing HA Protein of H5N1 Subtype Avian Influenza Virus and Analysis of Its Immunogenicity in Ducks

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