通过用重组人血清白蛋白-l7/l12（流产布鲁氏菌核糖体蛋白）和脂多糖联合疫苗接种来保护BALB/C小鼠免受流产布鲁氏菌544的攻击。

Protection of BALB/C mice against Brucella abortus 544 challenge by vaccination with combination of recombinant human serum albumin-l7/l12 (Brucella abortus ribosomal protein) and lipopolysaccharide.

作者信息

Pakzad Iraj, Rezaee Abbas, Rasaee Mohammad J, Hossieni Ahmad Zavaran, Tabbaraee Bahman, Kazemnejad Anoshirvan

机构信息

Department of Microbiology, Ilam University of Medical Sciences, Ilam, Iran.

出版信息

Roum Arch Microbiol Immunol. 2010 Jan-Mar;69(1):5-12.

PMID:21053778

Abstract

BACKGROUND

The immunogenic Brucella abortus ribosomal protein L7/L12 and Lipopolysaccharide (LPS) are promising candidate antigens for the development of subunit vaccines against brucellosis.

OBJECTIVE

This study was aimed to evaluate the protection of combination of recombinant HSA-L7/L12 fusion protein with LPS in Balb/c mouse.

MATERIALS AND METHODS

The recombinant HSA-L7/L12 fusion protein in Saccharomyces cerevisiae was expressed and purified by affinity chromatography column. LPS was extracted by n-butanol, purified by ultracentrifugation. BALB/c mouses were immunized in 9 groups with PBS, HSA, tHSA-L7/L12, L7/L12, LPS, LPS+ HSA, LPS+ tHSA-L7/L12, LPS+ L7/L12, B. abortus S19. ELISA, LTT tests and challenging two weeks after last injection were carried out. Bacterial count of spleen of immunized BALB/c mouse was done four weeks after challenging with virulent strain B. abortus 544.

RESULTS

In ELISA test the specific antibodies of tHSA-L7/L12 exhibited a dominance of immunoglobulin IgG1 over IgG2a. LPS-HSA and tHSA-L7/L12 + LPS produced a significantly higher antibody titer than LPS alone and L7/L12+LPS (P < 0.05). The predominant IgG subtype for LPS and L7/L12+LPS were IgG3. However, tHSA-L7/L12+ LPS and LPS+ HAS elicited predominantly IgG1 and IgG3 subtypes. In addition, the tHSA-L7/L12 fusion protein and L7/L12 elicited a strong T-cell proliferative response upon restimulation in vitro with recombinant tHSA-L7/L12 and L7/L12, suggesting the induction of a cellular immunity response in vivo. However, there was no significant difference proliferative response in L7/L12 and tHSA-L7/L12 fusion protein (P > 0.05). The combination of tHSA-L7/L12 fusion protein with LPS and B. abortus S19 induce higher level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice than other groups (P = 0.005).

CONCLUSIONS

The combination of tHSA-L7/L12 fusion protein with LPS had higher protective ability than LPS and fusion protein distinctly.

摘要

背景

具有免疫原性的流产布鲁氏菌核糖体蛋白L7/L12和脂多糖（LPS）是开发布鲁氏菌病亚单位疫苗的有前景的候选抗原。

目的

本研究旨在评估重组人血清白蛋白-L7/L12融合蛋白与LPS联合使用对Balb/c小鼠的保护作用。

材料与方法

在酿酒酵母中表达重组人血清白蛋白-L7/L12融合蛋白，并通过亲和层析柱进行纯化。用正丁醇提取LPS，通过超速离心进行纯化。将BALB/c小鼠分为9组，分别用PBS、人血清白蛋白、重组人血清白蛋白-L7/L12、L7/L12、LPS、LPS+人血清白蛋白、LPS+重组人血清白蛋白-L7/L12、LPS+L7/L12、流产布鲁氏菌S19株进行免疫。在最后一次注射后两周进行ELISA、淋巴细胞转化试验和攻毒试验。在用强毒株流产布鲁氏菌544攻毒四周后，对免疫的BALB/c小鼠脾脏进行细菌计数。

结果

在ELISA试验中，重组人血清白蛋白-L7/L12的特异性抗体显示免疫球蛋白IgG1占主导地位，高于IgG2a。LPS-人血清白蛋白和重组人血清白蛋白-L7/L12+LPS产生的抗体滴度明显高于单独的LPS和L7/L12+LPS（P<0.05）。LPS和L7/L12+LPS的主要IgG亚型为IgG3。然而，重组人血清白蛋白-L7/L12+LPS和LPS+人血清白蛋白主要诱导IgG1和IgG3亚型。此外，重组人血清白蛋白-L7/L12融合蛋白和L7/L12在用重组人血清白蛋白-L7/L12和L7/L12体外再刺激时引发了强烈的T细胞增殖反应，表明在体内诱导了细胞免疫反应。然而，L7/L12和重组人血清白蛋白-L7/L12融合蛋白的增殖反应没有显著差异（P>0.05）。重组人血清白蛋白-L7/L12融合蛋白与LPS的组合以及流产布鲁氏菌S19株在BALB/c小鼠中诱导的针对强毒株流产布鲁氏菌544攻毒的保护水平高于其他组（P = 0.005）。