摘要
【目的】探讨FliC蛋白作为黏膜佐剂对伪狂犬病病毒(Pseudorabies virus,PRV)Bartha-K61弱毒疫苗株的免疫增强作用,为PRV黏膜疫苗的研发奠定基础。【方法】将鼠伤寒沙门菌FliC基因克隆连接至原核表达载体pET-28a(+),连接产物转化大肠杆菌Trans10感受态细胞,提取质粒后进行双酶切鉴定并测序;将FliC-pET-28a转化大肠杆菌BL21(DE3)感受态细胞,诱导表达FliC蛋白,通过考马斯亮蓝染色和Western blotting对纯化后的FliC蛋白进行鉴定。设置Bartha-K61病毒液组和商品化Bartha-K61疫苗组作为对照,将FliC蛋白作为黏膜佐剂与Bartha-K61病毒液混合,滴鼻2次免疫BALB/c小鼠,测定免疫前(0 d)、免疫后7、14、21、28和35 d小鼠血清IgG和IgA水平,免疫后第35天对各组小鼠分别滴鼻PRV GD-WH病毒液10^(4.5) TCID_(50),攻毒后每日观察并记录小鼠生存状况。【结果】成功获得重组质粒FliC-pET-28a,实现了FliC蛋白的可溶性表达,纯化后的FliC蛋白大小约为58 ku。小鼠免疫试验结果显示,二免后Bartha-K61病毒液+FliC蛋白组血清IgG和IgA抗体均显著高于Bartha-K61病毒液组和商品化Bartha-K61疫苗组(P<0.05)。攻毒保护试验结果显示,Bartha-K61病毒液+FliC蛋白免疫能给小鼠提供100%的保护力,抵抗PRV GD-WH的致死性攻击,具有高于Bartha-K61病毒液单独免疫的保护力。【结论】FliC蛋白佐剂对PRV Bartha-K61弱毒疫苗株具有黏膜免疫增强作用,并使小鼠获得一定的免疫保护效力。
【Objective】This experiment aimed to investigate the immunopotentiating effect of FliC protein as a mucosal adjuvant on the Pseudorabies virus(PRV)Bartha-K61 attenuated vaccine strain,so as to lay a foundation for developing PRV-targeted mucosal vaccines.【Method】FliC gene was cloned from Salmonella Typhimurium and linked to the prokaryotic expression vector pET-28a(+),the linked product was transformed into Escherichia coli(E.coli)Trans10 competent cells,and the plasmid was extracted for double enzyme digestion identification and sequencing.FliC-pET-28a was transformed into E.coli BL21(DE3)competent cells to induce the expression of FliC protein,and the purified FliC protein was identified by Coomassie brilliant blue staining and Western blotting.Bartha-K61 virus suspension group and the commercial Bartha-K61 vaccine group were set up as control.FliC protein as mucosal adjuvant was mixed with Bartha-K61 virus solution,BALB/c mice were intranasally immunized twice.The levels of IgG and IgA in the serum of mice before immunization(day 0)and at post-immunization days 7,14,21,28 and 35 were measured.Mice in each group were challenged with PRV GD-WH of 10^(4.5) TCID_(50) on the 35th day after immunization and the survival was observed and recorded daily after challenge.【Result】The recombinant plasmid FliC-pET-28a was successfully obtained,the soluble expression of FliC was realized,and the size of the purified FliC protein was approximately 58 ku.The results of mouse immune test showed that the serum IgG and IgA antibodies of Bartha-K61 virus solution+FliC protein group were significantly higher than those of Bartha-K61 virus solution group and commercial Bartha-K61 vaccine group(P<0.05).The results of challenge protection test showed that Bartha-K61 virus solution+FliC protein could provide 100% protection to mice against the lethal attack of PRV GD-WH,which was higher than that of Bartha-K61 virus solution alone.【Conclusion】FliC protein adjuvant could enhance mucosal immunity of PRV Bartha-K61 attenuated vaccine strain,and enable mice to obtain certain immune protection.
作者
梁力荣
柯海意
臧莹安
翟少伦
康桦华
蒋智勇
李艳
张昆丽
周霞
勾红潮
李春玲
LIANG Lirong;KE Haiyi;ZANG Yingan;ZHAI Shaolun;KANG Huahua;JIANG Zhiyong;LI Yan;ZHANG Kunli;ZHOU Xia;GOU Hongchao;LI Chunling(Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China;Guangdong Key Laboratory of Livestock Disease Prevention,Guangdong Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Technology,Ministry of Agriculture Rural Affairs,Guangdong Provincial Enterprise Key Laboratory of Pig Raising and Swine Disease Control Technology,State Key Laboratory of Swine and Poultry Breeding Industry,Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;Gaozhou Agricultural School,Guangdong Province,Maoming 525200,China)
出处
《中国畜牧兽医》
北大核心
2025年第10期4864-4873,共10页
China Animal Husbandry & Veterinary Medicine
基金
广东省农业科学院协同创新中心项目(XTXM202202、XT202207)
猪禽种业全国重点实验室(2023QZ-NK13、GDNKY-ZQQZ-K25)
科技创新战略专项(高水平农科院建设)(R2021PY-QY006)
国家重点研发计划(2024YFD1800505)
地方猪种项目(2024-XBH-00-003)
省级现代农业产业体系创新团队建设项目(2024CXTD15)。
