巴斯德毕赤酵母多拷贝整合表达组装禽流感病毒样颗粒

doi:10.6048/j.issn.1001-4330.2021.11.022

新疆农业科学 ›› 2021, Vol. 58 ›› Issue (11): 2148-2156.DOI: 10.6048/j.issn.1001-4330.2021.11.022

• 畜牧兽医·草业 • 上一篇

巴斯德毕赤酵母多拷贝整合表达组装禽流感病毒样颗粒

薛倩¹^,²(), 马文戈²(), 沙依兰·卡依扎², 汪萍², 韩涛², 苗书魁², 夏俊², 陆桂丽²

1.新疆农业大学动物医学学院,乌鲁木齐 830000
2.新疆畜牧科学院兽医研究所/新疆畜牧科学院动物临床医学研究中心,乌鲁木齐 830011

收稿日期:2021-01-27 出版日期:2021-11-20 发布日期:2021-12-16
通信作者: 马文戈
作者简介:薛倩(1994-),女,新疆石河子人,硕士研究生,研究方向为动物病毒免疫学,(E-mail) 1607971717@qq.com
基金资助:
国家重点研发计划“家禽主要传染病综合防控研究及国际科技合作平台建设”(2016YFE0203200)

Avian Influenza Virus-like Particles Expressed and Assembled by Multi-copy Recombinant Pichia Pastoris

XUE Qian¹^,²(), MA Wenge²(), Shayilan Kayizha², WANG Ping², HAN Tao², MIAO Shukui², XIA Jun², LU Guili²

1. College of Animal Medicine, Xinjiang Agriculture University, Urumqi 830000, China
2. Institute of Veterinary Medicine, Xinjiang Academy of Animal Sciences, Urumqi 830011, China

Received:2021-01-27 Online:2021-11-20 Published:2021-12-16
Correspondence author: MA Wenge
Supported by:
National Key Research & Development Program of China "Prevention & Control of Domestic Fowl's Main Infectious Diseases and Platform Building of International Science & Technology Cooperation"(2016YFE0203200)

摘要/Abstract

摘要：

【目的】构建多拷贝整合表达禽流感病毒样颗粒的重组巴斯德毕赤酵母,为H5N1亚型禽流感基因工程疫苗提供基础。【方法】以巴斯德毕赤酵母GS115株18S rRNA基因(rDNA)部分序列,插入pPIC9K载体上XbaⅠ和Bsp1407Ⅰ位点间,构建多拷贝整合表达质粒p8K。再以禽流感病毒H5N1毒株基因组RNA为模板,RT-PCR扩增HA、M和NA基因,分别插入多拷贝载体p8K,构建表达质粒 p8K-HA/M/NA。表达质粒分别扩增后线性化,按比例混合,电转化GS115感受态细胞。增菌后,经遗传霉素G418抗性平板筛选、PCR鉴定携带HA、M和NA基因序列的多拷贝整合菌株。阳性菌株增菌、诱导表达、高压均质破碎后,Western-blot检测表达产物。【结果】PCR鉴定阳性的重组菌株,其细胞裂解蛋白,免疫印迹试验可检测到与HA、M₁和NA分子量一致的蛋白条带;电子显微镜可观察到大小约为80~120 nm 的病毒样颗粒,免疫鸡能产生抗禽流感病毒中和抗体。【结论】重组毕赤酵母能够多拷贝整合、表达、组装禽流感病毒样颗粒。

关键词: 禽流感病毒; 多拷贝整合; 病毒样颗粒

Abstract:

【Objective】 To construct multi-copy integrative pichia pastoris for expression and assembly of virus-like particles(VLPs) to support the research of genetically engineered vaccine of avian influenza virus(AIV) H5N1subtype. 【Method】 Partial 18S rRNA gene (rDNA) of Pichia pastoris GS115 strain was amplified out and inserted into enzymatic XbaⅠ and Bsp1407Ⅰ sites of plasmid pPIC9K to construct multi-copy integrative vector p8K. And, structural protein HA, M and NA genes of AIV H5N1 subtype were amplified by RT-PCR to construct expression plasmids p8K-HA/M/NA, respectively. Subsequently, those expression plasmids were linearized,, mixed proportionally and electro-transformed into Pichia pastoris GS115 strain, and screened on Geneticin G418 plates. Later, positive recombinants carrying multiple-copied HA, M and NA genes identified by PCR were picked out, enrichment cultured and induction expressed, harvested, broken by High speed homogenation and confirmed by Western-blot, respectively.【Result】 Cell lysates of positive recombinant yeasts confirmed by PCR method c be identified out by immunoblotting with three bands corresponding to HA, M₁ and NA. And, 80~120nm virus-like particles (VLPs) be observed by electron microscopy.chickens injected by VLPs produce specific neutralization antibodies against AIV.【Conclusion】 Recombinant pichia pastoris can multi-integrate, express and assemble AIV VLPs.

Key words: avian influenza virus; multi-copy integration; virus-like particles

中图分类号:

S813

薛倩, 马文戈, 沙依兰·卡依扎, 汪萍, 韩涛, 苗书魁, 夏俊, 陆桂丽. 巴斯德毕赤酵母多拷贝整合表达组装禽流感病毒样颗粒[J]. 新疆农业科学, 2021, 58(11): 2148-2156.

XUE Qian, MA Wenge, Shayilan Kayizha, WANG Ping, HAN Tao, MIAO Shukui, XIA Jun, LU Guili. Avian Influenza Virus-like Particles Expressed and Assembled by Multi-copy Recombinant Pichia Pastoris[J]. Xinjiang Agricultural Sciences, 2021, 58(11): 2148-2156.

图/表 7

表1 HA、M和NA基因扩增引物

Table 1 Primers for amplification of HA, M and NA genes

引物名称 Primers	引物序列(5’-3’) Sequences (5’-3’)	扩增长度(bp) Degree of amplification
HAF	GATATC(EcoRV)AAAATAGTGCTTCTTCTTGCA	1 704
HAR	GCGGCCGC(NotI)TTAAATGCAAATTCTGCATTG	1 704
MF	TACGTA(SnaBI)AGTCTTCTAACCGAGGTCGAA	982
MR	GCGGCCGC(Not I)TTACTCCAATTCTATGTTGAC	982
NAF	TACGTA(SnaBI)AATCCAAATCAGAAGATA	1 350
NAR	GCGGCCGC(Not I)CTACTTGTCAATGGTGAA	1 350

表2 免疫试验分组及剂量

Table 2 Groups and doses of immunization chickens

组别 Group	免疫分类 Immunologic classification	数量 Number (只)	接种剂量 Inoculation dose(μL)
A	重组蛋白组1	5	100
B	重组蛋白组2	5	200
C	重组蛋白组3	5	400
D	灭活疫苗组	5	500
E	酵母蛋白组	5	500
F	PBS对照组	5	500

图1 表达质粒限制性内切酶消化鉴定注:1.Gene Ruler 1Kb plus DNA Ladder,2. 质粒p8K-NA,3. 质粒p8K-M,4. 质粒p8K-HA,5. 质粒p8K,6. 质粒pPIC9K对照

Fig.1 Identification of expression plasmids digested by restriction endonuclease Note: 1. Gene Ruler 1Kb plus DNA Ladder, 2. plasmid p p8K-NA,3. plasmid p8K-M,4. plasmid p8K-HA,5. plasmid p8K,6. plasmid pPIC9K

图2 重组酵母PCR筛选与鉴定

Fig.2 Identification and screon of recombinant yeasts by PCR methar

图3 重组蛋白SDS-PAGE电泳与Western-blot检测注:1. 蛋白质Marker;2. GS115酵母蛋白;3. p8K重组GS115酵母蛋白;4. 禽流感病毒H5亚型抗原;5. 重组菌株rAI-a/1细胞碎片,6. 重组菌株rAI-a/1 30%硫酸铵沉淀破碎细胞上清

Fig.3 Recombinant proteins by SDS-PAGE and Western-blot Note: 1. Protein Marker; 2.Proteins of GS115; 3.Proteins of recombinant GS115 integrated by p8K; 4. Antigen of AIV H5 subtype; 5. Debris of the rAI-1/a; 6. Supernatant proteins precipitated by 30% (NH4)2 SO4 of the rAI-1/a

图4 电子显微镜观察VLPs

Fig.4 Observation of VLPs by electro-microscope

表3 重组蛋白血凝抑制抗体效价

Table 3 Titer of recombinant protein hemagglutination inhibition antibody

组别 Group	免疫分类 Immunologic classification	AIV H5亚型血凝抑制抗体效价
组别 Group	免疫分类 Immunologic classification	0 wps	1 wps	3 wps	6 wps	10 wps
A	重组VLPs组1	<2^-1	2^-2	2^-5	2^-4	2^-2
B	重组VLPs组2	<2^-1	2^-3	2^-6	2^-5	2^-3
C	重组VLPs组3	<2^-1	2^-3	2^-6	2^-6	2^-4
D	灭活疫苗组	<2^-1	2^-4	2^-8	2^-8	2^-6
E	酵母蛋白组	<2^-1	<2^-1	<2^-1	<2^-1	<2^-1
F	PBS组	<2^-1	<2^-1	<2^-1	<2^-1	<2^-1

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巴斯德毕赤酵母多拷贝整合表达组装禽流感病毒样颗粒

Avian Influenza Virus-like Particles Expressed and Assembled by Multi-copy Recombinant Pichia Pastoris

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