细粒棘球绦虫EgM123表面展示型酿酒酵母菌株的构建及免疫原性初步评价

doi:10.11843/j.issn.0366-6964.2025.01.035

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (1): 378-391.doi: 10.11843/j.issn.0366-6964.2025.01.035

细粒棘球绦虫EgM123表面展示型酿酒酵母菌株的构建及免疫原性初步评价

贾新月¹^,²(), 马静¹^,², 普娜¹^,², 赵文卿¹^,³, 陈旭珂¹^,², 张艳艳¹, 孙艳¹, 薄新文¹^,²^,*(), 王正荣¹^,*()

1. 省部共建绵羊遗传改良与健康养殖国家重点实验室/新疆农垦科学院畜牧兽医研究所, 石河子 832000
2. 石河子大学动物科技学院, 石河子 832000
3. 塔里木大学动物科学与技术学院, 阿拉尔 843300

收稿日期:2024-03-06 出版日期:2025-01-23 发布日期:2025-01-18
通讯作者: 薄新文,王正荣 E-mail:1342991830@qq.com;xinwen_bo@126.com;wzrtiger@sina.com
作者简介:贾新月(1997-)，女，辽宁朝阳人，硕士生，主要从事预防兽医学研究，E-mail：1342991830@qq.com
基金资助:
国家自然科学基金(32360887)；新疆生产建设兵团国际科技合作(2021BC008)；省部共建绵羊遗传改良与健康养殖国家重点实验室重大专项(2021ZD02)；兵团农业科技创新工程专项(NCG202213)联合资助

Construction and Evaluation of a Surface-displayed Saccharomyces cerevisiae EBY100/PYD1-EgM123 for Echinococcus granulosus

JIA Xinyue¹^,²(), MA Jing¹^,², PU Na¹^,², ZHAO Wenqing¹^,³, CHEN Xuke¹^,², ZHANG Yanyan¹, SUN Yan¹, BO Xinwen¹^,²^,*(), WANG Zhengrong¹^,*()

1. State Key Labaratory of Sheep Genetic Improvement and Healthy Production, Institute of Animal Husbandry and Veterinary, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China
2. College of Animal Science and Technology, Shehezi University, Shihezi 832000, China
3. College of Animal Science and Technology, Tarim University, Aral 843300, China

Received:2024-03-06 Online:2025-01-23 Published:2025-01-18
Contact: BO Xinwen, WANG Zhengrong E-mail:1342991830@qq.com;xinwen_bo@126.com;wzrtiger@sina.com

摘要/Abstract

摘要：

旨在构建并制备细粒棘球绦虫EBY100/pYD1-EgM123表面展示型酿酒酵母口服活载体菌株，并初步评价其免疫效果，以期为细粒棘球绦虫终末宿主酿酒酵母活载体口服疫苗的研发提供理论基础。克隆细粒棘球绦虫EgM123基因，构建EBY100/pYD1-EgM123表面展示型酿酒酵母菌株。诱导培养表面展示型酿酒酵母EBY100/pYD1-EgM123，并进行体外分析，通过免疫荧光分析以及BCA蛋白定量法对EgM123蛋白进行定位鉴定及定量分析。诱导表达表面展示型酿酒酵母EBY100/pYD1-EgM123菌株并免疫BALB/c小鼠。通过检测小鼠血清中抗体及细胞因子水平变化评价细粒棘球绦虫EgM123口服酵母活载体菌株的免疫效果。结果成功克隆了细粒棘球绦虫EgM123的ORF序列，长度为594 bp。PCR检测以及酶切鉴定结果表明，成功构建EBY100/pYD1-EgM123表面展示型酿酒酵母菌株。免疫荧光结果显示，EBY100/pYD1-EgM123表面具有绿色荧光，BCA蛋白定量法测定50 OD_{600 nm} EBY100/pYD1-EgM123诱导72 h后蛋白的表达量为15.59 μg，浓度为0.31 μg/OD_{600 nm}。抗体水平检测结果表明，EBY100/pYD1-EgM123免疫组特异IgG、IgA、IgM抗体的分泌均高于PBS组、EBY100组及EBY100/pYD1组(P < 0.05)。细胞因子检测发现，EBY100/pYD1-EgM123免疫组对小鼠细胞因子IL-17、IFN-γ的分泌量显著加强(P < 0.001)。综上所述，通过对细粒棘球绦虫EBY100/pYD1-EgM123表面展示型酿酒酵母口服活载体菌株免疫效果的初步评价，发现该口服活载体菌株具有免疫原性，可以刺激小鼠产生特异性免疫应答，进一步说明其具有成为终末宿主抗囊型包虫候选疫苗的潜力。

关键词: 细粒棘球绦虫, EgM123, 表面展示型酿酒酵母, 口服疫苗

Abstract:

The objective of this study is to construct a surface-displayed Saccharomyces cerevisiae EBY100/PYD1-EgM123 for Echinococcus granulosus, and to evaluate its immunogenicity. The EgM123 gene of E. granulosus was cloned, and a surface display yeast strain EBY100/PYD1-EgM123 was constructed. The S. cerevisiae strain EBY100/PYD1-EgM123 was examined using immunofluorescence assay, and BCA protein quantitative analysis to determine its localization and quantity. Additionally, experimental mice were immunized with the EBY100/pYD1-EgM123. The immune efficacy of the EBY100/pYD1-EgM123 was evaluated by measuring antibody and cytokine in the serum of the mice. The results showed that the ORF of EgM123 was successfully cloned and its length was 594 bp. The results of PCR and restriction endonuclease analysis showed that the surface-displayed S. cerevisiae EBY100/PYD1-EgM123 was successfully constructed. The results of immunofluorescence showed that the EBY100/PYD1-EgM123 protein exhibited green fluorescence on the surface of the S. cerevisiae. The expression level of EBY100/pYD1-EgM123 was quantified at 50 OD_{600 nm} using the BCA protein quantitative assay. At 72 h, the expression level was found to be 15.59 μg and 0.31 μg/OD_{600 nm}, respectively. The levels of specific IgG, IgA, and IgM in the EBY100/pYD1-EgM123 group were found to be significantly higher than those in the PBS Group, EBY100 Group, and EBY100/PYD1 group (P < 0.05). The results also indicated that the EBY100/PYD1-EgM123 effectively enhances the secretion of IL-17 and IFN-γ (P < 0.001). In conclusion, the present study successfully constructed the surface-displayed S. cerevisiae EBY100/PYD1-EgM123, and demonstrating its ability to stimulate specific immune responses in mice. It is further indicated that it has the potential to be a candidate vaccine against E. granulosus in the final host.

Key words: Echinococcus granulosus, EgM123, surface-displayed Saccharomyces cerevisiae, oral live vector vaccine

中图分类号:

S852.734

贾新月, 马静, 普娜, 赵文卿, 陈旭珂, 张艳艳, 孙艳, 薄新文, 王正荣. 细粒棘球绦虫EgM123表面展示型酿酒酵母菌株的构建及免疫原性初步评价[J]. 畜牧兽医学报, 2025, 56(1): 378-391.

JIA Xinyue, MA Jing, PU Na, ZHAO Wenqing, CHEN Xuke, ZHANG Yanyan, SUN Yan, BO Xinwen, WANG Zhengrong. Construction and Evaluation of a Surface-displayed Saccharomyces cerevisiae EBY100/PYD1-EgM123 for Echinococcus granulosus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 378-391.

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方案 Regimen	首次免疫 First immunity	加强免疫 Boost immunity
1	第1、2、3天每天灌服50 μL	第16、17、18天每天灌服50 μL
2	第1、2天每天灌服75 μL	第16、17天每天灌服75 μL
3	第1天灌服150 μL	第16天灌服150 μL

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细粒棘球绦虫EgM123表面展示型酿酒酵母菌株的构建及免疫原性初步评价

Construction and Evaluation of a Surface-displayed Saccharomyces cerevisiae EBY100/PYD1-EgM123 for Echinococcus granulosus

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