布鲁菌转录调节因子HFQ诱导机体免疫反应的分析

doi:10.11843/j.issn.0366-6964.2020.08.022

摘要/Abstract

摘要： 旨在分析布鲁菌（Brucella）转录调节因子HFQ诱导机体产生的免疫反应。以热灭活牛种布鲁菌S2308为模板，根据GenBank登录的S2308 hfq基因序列（BAB1_1134）设计引物，PCR扩增hfq基因片段后，将其克隆至原核表达载体pET-32a，转化大肠杆菌BL21（DE3）感受态细胞，诱导HFQ蛋白表达；利用SDS-PAGE电泳以及Western blot对重组HFQ蛋白（rHFQ）进行分析；pET-32a空载体、rHFQ和疫苗株M5-90刺激小鼠巨噬细胞RAW 264.7，利用ELISA试剂盒检测细胞因子IFN-γ和IL-4的表达水平；将pET-32a、rHFQ和M5-90免疫小鼠后，检测小鼠脾细胞中IFN-γ和IL-4的水平，以及小鼠血清中IgG抗体水平。结果显示，hfq基因大小为237 bp，编码79个氨基酸，rHFQ大约在25.8 ku处出现蛋白条带，纯化后为单一条带。Western blot结果显示，rHFQ具有较好的反应原性。rHFQ刺激RAW 264.7后，诱导IFN-γ和IL-4的水平与M5-90组相似，显著高于PBS组和pET-32a空载体组，且随着刺激时间的延长而升高。rHFQ免疫小鼠后，诱导脾细胞产生IFN-γ和IL-4的水平，小鼠血清中IgG的水平与M5-90组相似，显著高于PBS组和pET-32a空载体组。布鲁菌HFQ蛋白具有较好的反应原性，并能诱导机体产生较高的细胞免疫和体液免疫水平，是布鲁菌亚单位疫苗研制较理想的候选抗原。

关键词: 布鲁菌, HFQ, 免疫反应

Abstract: This experiment was conducted to study immune responses induced by Brucella transcriptional regulatory factor HFQ. In this study, a pair of primers were designed according to hfq gene sequence of B. abortus S2308 from GenBank (BAB1_1134), and the heat inactivated S2308 was used as template to amplify hfq gene by PCR. And then the hfq gene was cloned into pET-32a vector and transformed to E. coli BL21 (DE3) and induced the expression of recombinant HFQ protein (rHFQ). The expressed proteins were analyzed by SDS-PAGE and Western blot. Expression levels of cytokines IFN-γ and IL-4 in murine macrophages (RAW 264.7) stimulated with pET-32a empty plasmid, rHFQ and current vaccine M5-90 were detected by ELISA. Mice were immunized with pET-32a empty plasmid, rHFQ and current vaccine M5-90, and IFN-γ, IL-4 in splenocytes and IgG antibody levels in serum of mice were detected by ELISA. The results showed that the full length of hfq gene was 237 bp, encoding 79 amino acids. rHFQ was approximately 25.8 ku by the SDS-PAGE. There was a single band after purification. Results of Western blot indicated that the rHFQ had good reactogenicity. When RAW 264.7 cells were stimulated with pET-32a, rHFQ and M5-90, the levels of IFN-γ and IL-4 in rHFQ group were similar to those in M5-90 group, significantly higher than those in PBS group and pET-32a empty plasmid control group. The levels of IFN-γ and IL-4 were increased over time. When mice were immunized with pET-32a, rHFQ and M5-90, the levels of IFN-γ and IL-4 in splenocytes and IgG antibody levels in serum in rHFQ group were similar to those in M5-90 group, significantly higher than those in PBS group and pET-32a empty plasmid control group. The HFQ protein of Brucella has good reactivity and can induce the body to produce high levels of cellular and humoral immunity. It is an ideal candidate antigen for the development of Brucella subunit vaccines.

Key words: Brucella, HFQ, Immune responses

中图分类号:

王书利, 张慧茹, 毕艳琪, 王德娟, 陈琳, 张晓婷, 李志强. 布鲁菌转录调节因子HFQ诱导机体免疫反应的分析[J]. 畜牧兽医学报, 2020, 51(8): 1977-1984.

WANG Shuli, ZHANG Huiru, BI Yanqi, WANG Dejuan, CHEN Lin, ZHANG Xiaoting, LI Zhiqiang. Analysis of Immune Responses Induced by Brucella Transcriptional Regulatory Factor HFQ[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1977-1984.

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