猪轮状病毒VP4基因重组腺病毒的构建及抗体水平评价

doi:10.16431/j.cnki.1671-7236.2023.01.026

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (1): 260-269.doi: 10.16431/j.cnki.1671-7236.2023.01.026

猪轮状病毒VP4基因重组腺病毒的构建及抗体水平评价

肖莉^1,2,3, 牛小杰^1,2,3, 刘庆庆^1,2,3, 王月丽^1,2,3, 陈创夫^1,2,3, 易继海^1,2,3

1. 石河子大学动物科技学院, 石河子 832000;
2. 绵羊健康养殖与人兽共患病防控协同创新中心, 石河子 832000;
3. 新疆生产建设兵团动物疾病防控重点实验室, 石河子 832000

收稿日期:2022-04-19 出版日期:2023-01-05 发布日期:2023-01-04
通讯作者: 陈创夫, 易继海 E-mail:ccf-xb@163.com;724050645@qq.com
作者简介:肖莉,E-mail:379293804@qq.com。
基金资助:
西部地区高发人兽共患传染病性疾病防治协同创新中心（2013-179）

Construction of Recombinant Adenovirus with VP4 Gene of Porcine Rotavirus and Evaluation of Antibody Level

XIAO Li^1,2,3, NIU Xiaojie^1,2,3, LIU Qingqing^1,2,3, WANG Yueli^1,2,3, CHEN Chuangfu^1,2,3, YI Jihai^1,2,3

1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
2. Collaborative Innovation Center for Healthy Sheep Breeding and Zoonotic Disease Prevention and Control, Shihezi 832000, China;
3. Key Laboratory of Animal Disease Prevention and Control, Xinjiang Production and Construction Corps, Shihezi 832000, China

Received:2022-04-19 Online:2023-01-05 Published:2023-01-04

摘要/Abstract

摘要： 【目的】构建猪轮状病毒（Porcine rotavirus，PoRV）流行株PoRV G9P[23]型的VP4基因重组腺病毒，为开发PoRV候选基因工程疫苗奠定基础。【方法】参考GenBank中流行株PoRV G9P[23]型的VP4基因序列（登录号：MH898990.1）合成PoRV VP4基因，将得到的目的基因与腺病毒穿梭载体pAdTrack-CMV进行重组，转化大肠杆菌Top10感受态细胞，构建腺病毒穿梭载体pAdTrack-CMV-VP4；腺病毒穿梭载体经过Pme Ⅰ内切酶线性化处理后与含有腺病毒骨架pAdEasy-1的大肠杆菌BJ5183感受态细胞进行同源重组获得重组质粒pAd-VP4，对重组质粒进行Pac Ⅰ酶切鉴定，并转化大肠杆菌DH5α感受态细胞。将重组质粒转染HEK293A细胞获得重组腺病毒rAd-VP4，对该重组腺病毒进行扩大培养并测定重组腺病毒的半数组织培养感染剂量（TCID₅₀）；通过RT-PCR检测其体外表达情况，Western blotting检测其反应原性；将制备的重组腺病毒用不同病毒滴度和不同免疫次数对小鼠进行腹腔免疫，收集血清通过ELISA法测定IgG抗体水平。【结果】RT-PCR扩增出1条大小为2 343 bp的rAd-VP4重组腺病毒条带，测序结果正确，表明重组腺病毒rAd-VP4构建成功，测得rAd-VP4病毒滴度为10^6.5 TCID₅₀，Western blotting结果表明，重组腺病毒rAd-VP4在蛋白水平上得到了正确表达，蛋白的分子质量约为87 ku。小鼠IgG抗体检测结果表明，在用10^6.5 TCID₅₀ rAd-VP4免疫后的第35和42天，小鼠血清中的IgG抗体水平显著高于10^5.3 TCID₅₀ TGE-PED-PRV三联活疫苗IgG抗体水平（P<0.05）；10^6.5 TCID₅₀ rAd-VP4在免疫后第35和42天产生的抗体水平显著高于10^5.5和10^4.5 TCID₅₀ rAd-VP4（P<0.05），而10^5.5 TCID₅₀ rAd-VP4在免疫后第28天产生的抗体水平显著高于10^6.5和10^4.5 TCID₅₀ rAd-VP4（P<0.05）。10^6.5 TCID₅₀ rAd-VP4的2次免疫和3次免疫产生的IgG抗体在不同免疫时间均差异不显著（P>0.05）。【结论】本研究成功构建了重组腺病毒rAd-VP4，其病毒滴度为10^6.5 TCID₅₀。10^6.5和10^5.5 TCID₅₀ rAd-VP4分别在第42和28天产生较高的IgG抗体水平，2次免疫和3次免疫对产生IgG抗体水平均无显著影响。试验结果可为开发PoRV重组腺病毒候选疫苗提供参考。

关键词: 猪轮状病毒(PoRV); 重组腺病毒载体; IgG抗体水平

Abstract: 【Objective】 The purpose of this study was to construct a recombinant Adenovirus with VP4 gene of Porcine rotavirus(PoRV) epidemic strain PoRV G9P[23] in China, and lay a preliminary foundation for the development of candidate engineering vaccine of PoRV.【Method】 PoRV VP4 gene was synthesized by referring to the VP4 gene sequence of PoRV G9P[23] strain in GenBank (accession No.:MH898990.1). The obtained target gene was recombined with Adenovirus shuttle vector pAdTrack-CMV and transferred into Escherichia coli Top10 competent cells to construct Adenovirus shuttle vector pAdTrack-CMV-VP4. The recombinant plasmid pAd-VP4 was obtained by homologous recombination with BJ5183 competent cells containing adenovirus backbone pAdEasy-1 after linearized by PmeⅠ endonuclease. The recombinant plasmid was digested by PacⅠ enzyme and transformed into Escherichia coli DH5α competent cells. The recombinant plasmid was transfected into HEK293A cells to obtain recombinant Adenovirus rAd-VP4. The recombinant Adenovirus was expanded and the half tissue culture infective dose (TCID₅₀) of recombinant Adenovirus was determined.RT-PCR was used to detect its expression in vitro, and its reactivity was detected by Western blotting. The prepared recombinant Adenovirus was inoculated mice intraperitoneally with different virus titers and different times of immunization. The serum was collected to determine IgG antibody levels by ELISA kit.【Result】 A recombinant Adenovirus rAd-VP4 with a size of 2 343 bp was amplified by RT-PCR, and the sequencing results were correct, indicating that the recombinant Adenovirus rAd-VP4 was successfully constructed. The viral titer of rAd-VP4 was 10^6.5 TCID₅₀. The recombinant Adenovirus rAd-VP4 was correctly expressed at the protein level and the molecular mass of the protein was about 87 ku. The results of antibody detection showed that the serum IgG antibody level of mice immunized with 10^6.5 TCID₅₀ rAd-VP4 was significantly higher than that of 10^5.3 TCID₅₀ TGE-PED-PRV triple live vaccine on the 35th and 42nd day(P<0.05). The antibody level of 10^6.5 TCID₅₀ rAd-VP4 on the 35th and 42nd day after immunization was significantly higher than that of 10^5.5 and 10^4.5 TCID₅₀ rAd-VP4 (P<0.05). The antibody level of 10^5.5 TCID₅₀ rAd-VP4 on the 28th day after immunization was significantly higher than that of 10^6.5 and 10^4.5 TCID₅₀ rAd-VP4 (P<0.05). There was no significant difference in IgG antibody produced by 10^6.5 TCID₅₀ rAd-VP4 immunizing 2 and 3 times at different immunization times (P>0.05).【Conclusion】 In this study, the recombinant Adenovirus rAd-VP4 was successfully constructed, and it's virus titer was 10^6.5 TCID₅₀. 10^6.5 and 10^5.5 TCID₅₀ rAd-VP4 produced higher IgG antibody levels on the 42th and 28th day, respectively. There was no significant difference in IgG antibody level between the second immunization and the third immunization. The results could provide reference for the development of PoRV recombinant Adenovirus vaccine candidate.

Key words: Porcine rotavirus(PoRV); recombinant Adenovirus vector; IgG antibody level

中图分类号:

S858.28

肖莉, 牛小杰, 刘庆庆, 王月丽, 陈创夫, 易继海. 猪轮状病毒VP4基因重组腺病毒的构建及抗体水平评价[J]. 中国畜牧兽医, 2023, 50(1): 260-269.

XIAO Li, NIU Xiaojie, LIU Qingqing, WANG Yueli, CHEN Chuangfu, YI Jihai. Construction of Recombinant Adenovirus with VP4 Gene of Porcine Rotavirus and Evaluation of Antibody Level[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 260-269.

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猪轮状病毒VP4基因重组腺病毒的构建及抗体水平评价

Construction of Recombinant Adenovirus with VP4 Gene of Porcine Rotavirus and Evaluation of Antibody Level

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