牛肠病毒VP1基因重组腺病毒的构建及其对小鼠的免疫原性评价

doi:10.11843/j.issn.0366-6964.2025.09.039

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4615-4625.doi: 10.11843/j.issn.0366-6964.2025.09.039

牛肠病毒VP1基因重组腺病毒的构建及其对小鼠的免疫原性评价

马思琪^1,2, 吕雯雯^1,2, 陈俊贞^1,2, 李建林^1,2, 刘昱成³, 关团⁴, 丁剑⁴, 刘浩然¹, 叶鸿艳¹, 杨莉^1,2, 付强^1,2*, 史慧君^1,2*

1. 新疆农业大学动物医学学院, 乌鲁木齐 830052;
2. 新疆草食动物新药研究与创制自治区重点实验室, 乌鲁木齐 830052;
3. 省部共建绵羊遗传改良与健康养殖国家重点实验室, 石河子 832000;
4. 新疆维吾尔自治区动物疫病预防控制中心, 乌鲁木齐 831599

收稿日期:2024-09-06 发布日期:2025-09-30
通讯作者: 付强，主要从事病原微生物致病机制研究，E-mail：fq198505@gmail.com；史慧君，主要从事抗病毒药物研究，E-mail：shihuijunmm@163.com
作者简介:马思琪(1999-)，女，新疆乌鲁木齐人，硕士生，主要从事病原微生物致病机制研究，E-mail：kkkk0818kk@163.com；吕雯雯(2000-)，女，河南商丘人，硕士生，主要从事病原微生物致病机制研究，E-mail：930014404@qq.com
基金资助:
国家自然科学基金地区基金项目(32160829;32260881)；国家自然科学基金地区科学基金项目(32460879)；自治区研究生科研创新项目(XJ2024G128)；新疆维吾尔自治区兽医学特色学科和“新疆草食动物新药研究与创制”重点实验室开放课题(XJCDVM-HDRC-S202411)

Construction of Recombinant Adenovirus with Bovine Enterovirus VP1 Gene and Evaluation of Its Immunogenicity in Mice

MA Siqi^1,2, Lü Wenwen^1,2, CHEN Junzhen^1,2, LI Jianlin^1,2, LIU Yucheng³, GUAN Tuan⁴, DING Jian⁴, LIU Haoran¹, YE Hongyan¹, YANG Li^1,2, FU Qiang^1,2*, SHI Huijun^1,2*

1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China;
2. Key Laboratory of New Drug Research and Creation for Herbivorous Animals, Xinjiang Autonomous Region, Urumqi 830052, China;
3. State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding Jointly Established by the Ministry and the Province, Shihezi 832000, China;
4. Animal Disease Prevention and Control Center of Xinjiang Uygur Autonomous Region, Urumqi 831599, China

Received:2024-09-06 Published:2025-09-30

摘要/Abstract

摘要： 构建表达牛肠病毒(bovine enterovirus，BEV)结构蛋白VP1的重组腺病毒，并对其进行免疫原性和安全性的初步验证。首先，应用PCR扩增BEV VP1基因，并将其克隆至腺病毒载体pDC316-CMV-copGFP，获得的重组质粒pDC316-VP1-copGFP与pBHGlox-△E1,3cre共转染至293细胞后，进行病毒滴度测定及RT-PCR鉴定；其次，构建pET-32a-BEV-VP1原核表达质粒，优化诱导时间和温度后纯化BEV VP1蛋白；同时，将阴性对照腺病毒rAdv-copGFP、重组腺病毒rAdv-VP1及空白对照PBS分别肌注免疫BALB/c小鼠，观察小鼠临床症状；随后于免疫21 d后感染BEV，并于攻毒第0、5、10、15天采集小鼠肝、肺、脾、肾、小肠，分别提取组织RNA并制作病理切片，检测各组织中病毒载量并观察组织病理变化；最后，使用ELISA检测免疫血清中总IgG抗体、特异性抗体、细胞因子IFN-γ和IL-4水平。结果显示：成功构建腺病毒载体pDC316-VP1-copGFP；筛选出最优VP1原核表达条件为37 ℃诱导8 h，成功纯化出pET-32a-BEV-VP1蛋白；腺病毒免疫小鼠血清能够识别纯化后的VP1蛋白；重组腺病毒免疫能使小鼠产生特异性抗体，从而提高体液免疫与细胞免疫水平。本研究成功构建表达BEV VP1蛋白的重组腺病毒，在免疫小鼠后能够较好地诱导小鼠体内产生体液免疫应答和细胞免疫应答，从而对BEV感染起到了一定的保护作用，为进一步研发BEV新型疫苗奠定基础。

关键词: 牛肠病毒, 重组腺病毒, 结构蛋白VP1, 细胞免疫

Abstract: This study aimed to construct a recombinant adenovirus expressing the structural protein VP1 of bovine enterovirus (BEV), and to perform preliminary verification of its immunogenicity and safety. The BEV VP1 gene was amplified by PCR and cloned into the adenoviral vector pDC316-CMV-copGFP. The recombinant plasmid pDC316-VP1-copGFP obtained was co-transfected with pBHGlox-△E1,3cre into 293 cells, and then virus titer assay and RT-PCR were carried out to identify the virus; the construction of pET-32a-BEV-VP1 prokaryotic expression plasmid was constructed and BEV VP1 protein was purified after optimising the induction time and temperature; negative control adenovirus rAdv-copGFP, recombinant adenovirus rAdv-VP1, and blank control PBS were used to immunise BALB/c mice by myocardial injection to observe the clinical symptoms of mice; mice were infected with BEV after 21 days of immunisation and liver, spleen, lung, spleen and blood samples were collected from mice on day 0, 5, 10, and 15. The samples were collected from mice on day 0, 5, 10 and 15. After 21 days of BEV infection, the liver, lung, spleen, kidney and small intestine were collected on day 0, 5, 10 and 15. Tissue RNA was extracted and pathological sections were made, and the viral load in each tissue was detected and the histopathological changes were observed; the serum of adenovirus-immunised mice was tested for the response to the prokaryotic expression of the VP1 protein by Western blot; and the levels of total IgG antibody, specific antibody, and cytokine IFN-γ, IL-4 in the immunised serum were detected by ELISA. The adenoviral vector pDC316-VP1-copGFP was successfully constructed; the optimal conditions for VP1 prokaryotic expression were induced at 37 ℃ for 8 h, and pET-32a-BEV-VP1 protein was successfully purified; the serum of adenoviral-immunised mice was able to recognise the purified VP1 protein; the recombinant adenoviral immunisation was able to make the mice produce specific antibodies, which increased the levels of humoral immunity and cellular immunity. The recombinant adenovirus immunisation can make mice produce specific antibodies, thus improving humoral and cellular immunity. The recombinant adenovirus expressing BEV VP1 protein was successfully constructed in this study, and it could induce humoral and cellular immune responses in mice after immunisation, thus exerting a certain degree of protection against BEV infection, and laying a foundation for the further development of a new BEV vaccine.

Key words: bovine enterovirus, recombinant adenovirus, structural protein VP1, cellular immunity

中图分类号:

马思琪, 吕雯雯, 陈俊贞, 李建林, 刘昱成, 关团, 丁剑, 刘浩然, 叶鸿艳, 杨莉, 付强, 史慧君. 牛肠病毒VP1基因重组腺病毒的构建及其对小鼠的免疫原性评价[J]. 畜牧兽医学报, 2025, 56(9): 4615-4625.

MA Siqi, Lü Wenwen, CHEN Junzhen, LI Jianlin, LIU Yucheng, GUAN Tuan, DING Jian, LIU Haoran, YE Hongyan, YANG Li, FU Qiang, SHI Huijun. Construction of Recombinant Adenovirus with Bovine Enterovirus VP1 Gene and Evaluation of Its Immunogenicity in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4615-4625.

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牛肠病毒VP1基因重组腺病毒的构建及其对小鼠的免疫原性评价

Construction of Recombinant Adenovirus with Bovine Enterovirus VP1 Gene and Evaluation of Its Immunogenicity in Mice

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