旋毛虫14-3-3蛋白的定位及免疫效果分析

doi:10.11924/j.issn.1000-6850.casb2024-0395

中国农学通报 ›› 2025, Vol. 41 ›› Issue (8): 123-132.doi: 10.11924/j.issn.1000-6850.casb2024-0395

• 畜牧·动物医学·蚕·蜂 • 上一篇下一篇

旋毛虫14-3-3蛋白的定位及免疫效果分析

钱艳红¹(), 温肖会¹, 宋帅¹, 牛瑞辉¹, 吕殿红¹, 郑博彬¹, 袁子国², 罗胜军¹()

¹ 广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站，广州 510640
² 华南农业大学兽医学院，广州 510642

收稿日期:2024-06-21 修回日期:2024-11-19 出版日期:2025-03-15 发布日期:2025-03-14
通讯作者:
罗胜军，男，1974年出生，安徽望江人，副研究员，硕士，研究方向：动物疫病防控与监测研究。通信地址：510640 广东省广州市天河区五山白石岗，Tel：020-85290911，E-mail：422368881@qq.com。
作者简介:
钱艳红，女，1995年出生，安徽宿州人，科研辅助人员，硕士，研究方向：兽医寄生虫与寄生虫病学。通信地址：510640 广东省广州市天河区五山白石岗，Tel：020-85291469，E-mail：3538937727@qq.com。
基金资助:
广东省基础与应用基础研究基金项目“cAMP对副猪嗜血杆菌相关生物学功能的影响研究”(2022A1515012239)

Localization and Immune Efficacy Analysis of 14-3-3 Protein in Trichinella spiralis

QIAN Yanhong¹(), WEN Xiaohui¹, SONG Shuai¹, NIU Ruihui¹, LV Dianhong¹, ZHENG Bobin¹, YUAN Ziguo², LUO Shengjun¹()

¹ Institute of Animal Health, Guangdong Academy of Agricultural Sciences/ Guangdong Province Key Laboratory of Livestock Disease Prevention /Guangdong Scientific Observation and Experimental Station for Veterinary Drugs and Diagnostic Technologies, Ministry of Agriculture and Rural Affairs, Guangzhou 510640
² College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642

Received:2024-06-21 Revised:2024-11-19 Published:2025-03-15 Online:2025-03-14

摘要/Abstract

摘要：

为了获得旋毛虫(T1)14-3-3蛋白，并对其生物学特征鉴定评估。使用相关网站及软件对旋毛虫14-3-3(Ts14-3-3)蛋白生物学信息进行分析预测；利用基因克隆和原核表达技术将Ts14-3-3与pET-28a进行重组，并获得重组Ts14-3-3(rTs14-3-3)蛋白，经Western blot鉴定其抗原性后，制备rTs14-3-3蛋白免疫血清，并采用qPCR技术和间接免疫荧光技术鉴定其在不同虫期的表达及肌幼虫中的定位；通过动物实验评估rTs14-3-3蛋白对旋毛虫感染小鼠肌肉中幼虫的减虫效果。成功制备出可溶性的rTs14-3-3蛋白，大小约为33 kDa，并能够特异性识别猪旋毛虫阳性血清，制备的免疫血清效价高达1:512000，该蛋白在旋毛虫肌幼虫内部大量表达，基因在旋毛虫肠道感染期幼虫、成虫、肌幼虫中均有转录，对小鼠肌幼虫减虫率为55.2%。rTs14-3-3蛋白的反应原性和免疫原性良好，能够减少旋毛虫感染小鼠组织中的肌幼虫数量，具有作为血清学诊断和候选疫苗分子抗原的潜力。

关键词: 旋毛虫, 旋毛虫14-3-3蛋白, 生物信息学, 原核表达, 抗原性分析, 免疫保护

Abstract:

To obtain the 14-3-3 protein of Trichinella spiralis (T1) and to identify and evaluate its biological characteristics, the biological information of 14-3-3(Ts14-3-3) protein of Trichinella spiralis was analyzed and predicted using relevant websites and software. Ts14-3-3 was recombined with pET-28a using gene cloning and prokaryotic expression techniques, and the recombinant Ts14-3-3(rTs14-3-3) protein was obtained. After the antigenicity was identified by Western blot, the rTs14-3-3 protein immune serum was prepared. qPCR and indirect immunofluorescence were used to identify its expression in different insect stages and its localization in muscle larvae. The effect of rTs14-3-3 protein on larvae in the muscle of mice infected with Trichinella spiralis was evaluated by animal experiments. Soluble rTs14-3-3 protein was successfully prepared, with a size of about 33 kDa, and it could specifically recognize the positive serum of porcine Trichinella spiralis. The titer of the prepared immune serum was as high as 1:52000. The protein was highly expressed in the muscle larvae of Trichinella spiralis, and the gene was transcribed in the larvae, adults, and muscle larvae during the intestinal infection stage of Trichinella spiralis. The pest reduction rate of mouse muscle larvae was 55.2%. rTs14-3-3 protein has good reactivity and immunogenicity, can reduce the number of muscle larvae in mouse tissues infected by Trichinella spiralis, and has potential as a serological diagnosis and candidate vaccine molecular antigen.

Key words: Trichinella spiralis, Trichinella spiralis 14-3-3 protein, bioinformatics, prokaryotic expression, antigenic analysis, immune protection

钱艳红, 温肖会, 宋帅, 牛瑞辉, 吕殿红, 郑博彬, 袁子国, 罗胜军. 旋毛虫14-3-3蛋白的定位及免疫效果分析[J]. 中国农学通报, 2025, 41(8): 123-132.

QIAN Yanhong, WEN Xiaohui, SONG Shuai, NIU Ruihui, LV Dianhong, ZHENG Bobin, YUAN Ziguo, LUO Shengjun. Localization and Immune Efficacy Analysis of 14-3-3 Protein in Trichinella spiralis[J]. Chinese Agricultural Science Bulletin, 2025, 41(8): 123-132.

图/表 19

图1. ProtScale在线网站预测Ts14-3-3蛋白亲疏水性 Ts14-3-3蛋白结构中既有亲水性氨基酸也有疏水性氨基酸，但亲水性氨基酸所占比例较高，因此该蛋白表现为亲水性。

图2. SignalP 5.0在线网站预测Ts14-3-3蛋白信号肽 Ts14-3-3蛋白氨基酸序列中无信号肽。

图3. TMHMM-2.0在线网站预测Ts14-3-3蛋白跨膜结构 Ts14-3-3蛋白无跨膜结构，为膜外蛋白

图4. PSIPRED Workbench在线网站预测Ts14-3-3蛋白二级结构 Ts14-3-3蛋白二级结构包括螺旋结构和无规则卷曲结构

图5. SWISS-MODEL在线网站预测Ts14-3-3蛋白三级结构

图6. DNAstar分析软件预测Ts14-3-3蛋白抗原位点

图7. NJ邻接法(Neighbour Joining)构建Ts14-3-3系统发育进化树

图8. RT-PCR扩增 M：DL 2000 DNA Marker；1：阴性对照；2~3：Ts14-3-3基因RT-PCR扩增产物

图9. 原核重组表达载体PCR鉴定 M：DL 2000 DNA Marker；1：阴性对照；2~3：pET-28a-Ts14-3-3菌液

图10. rTs14-3-3蛋白可溶性探究蛋白凝胶电泳 M：180 kDa预染蛋白质Marker；1：诱导后的pET-28a空载菌体超声后上清；2：诱导后的pET-28a-Ts14-3-3重组菌体超声后上清；3：诱导后的pET-28a空载菌体超声后沉淀；4：诱导后的pET-28a-Ts14-3-3重组菌体超声后沉淀

图11. 不同温度诱导重组蛋白表达蛋白凝胶电泳 M：180 kDa预染蛋白质Marker；1：诱导后的PET-28空载菌液；2：未诱导的pET-28a-Ts14-3-3重组菌液；3~6：不同温度下诱导的pET-28a-Ts14-3-3重组菌液（1 mM IPTG，温度分别为：16、25、30、37℃，诱导12 h）

图12. 不同浓度IPTG诱导重组蛋白表达蛋白凝胶电泳 M：180 kDa预染蛋白质Marker；1：未诱导的PET-28空载菌液；2：未诱导的pET-28a-Ts14-3-3重组菌液； 3~10：不同浓度IPTG诱导的pET-28a-Ts14-3-3重组菌液（IPTG浓度分别为：0.1、0.3、0.5、0.7、0.9、1.1、1.3、1.5 mM，30℃诱导12 h）

图13. 不同时间诱导重组蛋白表达蛋白凝胶电泳 M：180 kDa预染蛋白质Marker；1~7：诱导的pET-28a-Ts14-3-3重组菌液（0.3 mM IPTG，30℃分别诱导：2、4、6、8、10、12、14 h）

图14. 重组蛋白纯化蛋白凝胶电泳 M：180 kDa预染蛋白质Marker；1：诱导后的pET-28a-Ts14-3-3重组菌体超声后上清；2~4：洗脱液1~3

图15. Western blot鉴定 M：180 kDa预染蛋白质Marker；A：1：猪旋毛虫阴性血清作一抗孵育；B：1~2：猪旋毛虫阳性血清作一抗孵育

图16. ELISA方法效价评估

图17. 实时荧光定量PCR方法检测Ts14-3-3基因相对表达水平

图18. 旋毛虫肌肉幼虫(ML)冷冻切片Ts14-3-3免疫荧光定位 A：孵育一抗为兔Ts14-3-3重组蛋白免疫血清；B：孵育一抗为兔阴性对照血清

图19. 旋毛虫肌幼虫减虫率分析

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旋毛虫14-3-3蛋白的定位及免疫效果分析

Localization and Immune Efficacy Analysis of 14-3-3 Protein in Trichinella spiralis

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