布鲁氏菌外膜蛋白OMP25 诱导BALB/ c 小鼠免疫反应的分析
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作者单位:

1. 河南科技大学动物科技学院,河南 洛阳 471003;2. 商丘师范学院生物与食品学院,河南 商丘 476000


Immune responses of BALB / c induced by Brucella melitensis outer membrane protein OMP25
Author:
Affiliation:

1. College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China.2. College of Biology and Food, Shangqiu Normal University, Shangqiu 476000

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    摘要:

    目的 本研究旨在分析布鲁氏菌外膜蛋白OMP25 诱导机体产生的免疫反应。方法 采用PCR 方法扩增羊种布鲁氏菌16M 的omp25 基因,克隆至pET-32a 质粒。重组质粒pET32a-OMP25 转化至大肠杆菌BL21(DE3)中,并诱导表达。利用Ni2+柱纯化试剂盒进行蛋白纯化。用纯化的重组蛋白(rOMP25)和Rev. 1 疫苗株免疫小鼠,检测小鼠脾细胞中IFN-γ 和IL-2 的水平,以及小鼠血清中IgG 抗体和抗炎因子IL-10 水平,并用Western Blot检测反应原性。结果 (1)omp25 基因大小为642 bp,编码214 个氨基酸,rOMP25 大约在40. 8 × 103 处出现蛋白条带,纯化后为单一条带。(2)用t 检验方法统计显示,rOMP25 免疫小鼠后,诱导脾细胞产生IFN-γ 和IL-2 的水平,小鼠血清中IgG 和IL-10 的水平与Rev. 1 组相似,显著高于对照组( P < 0. 01)。(3)Western Blot 检测结果显示,rOMP25 具有较好的反应原性。结论 rOMP25 具有较好的免疫原性和反应原性,为进一步研究OMP25 蛋白的功能,以及研制相关疫苗提供了生物材料。

    Abstract:

    Objective The purpose of this study was to analyze the immune responses induced by Brucellamelitensis(B. melitensis) outer membrane protein OMP25. Methods The omp25 gene from B. melitensis 16M was amplifiedby PCR. The fragment was cloned into pET-32a vector plasmid. The constructed recombinant plasmid pET32a-OMP25 wastransformed to E. coli BL21 (DE3) and was induced to express the fusion protein. Then the protein was purified using aNi2+ column purification kit. Mice were immunized with rOMP25 and current vaccine Rev. 1, and IFN-γ, IL-2 insplenocytes and IgG antibody and anti-inflammatory factor IL-10 levels in the serum were detected by ELISA. Thereactionogenicity was detected by Western Blot. Results (1) The full length of the omp25 gene was 642 bp, encoding 214amino acids. rOMP25 was approximately 40. 8 × 103 as measured by SDS-PAGE. There was a single band after purification. (2) The t-test method showed that mice were immunized with rOMP25 and Rev. 1, the levels of IFN-γ andIL-2 in splenocytes, IgG antibody and IL-10 levels in serum in the rOMP25 group were similar to those in the Rev. 1 group,and significantly greater than those in the control ( P < 0. 01). (3) rOMP25 had good reactivity as observed in the WestemBlot. These result confirmed that rOMP25 could induce the body to produce high levels of cellular and humoral immunity,with good reactivity. Conclusions This study provides technical support for further study of the function of OMP25 proteinfor B. melitensis related vaccine development.

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王书利,李志强,魏淑娟,司丽芳.布鲁氏菌外膜蛋白OMP25 诱导BALB/ c 小鼠免疫反应的分析[J].中国实验动物学报,2022,30(5):655~661.

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  • 收稿日期:2021-10-21
  • 在线发布日期: 2023-04-13
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