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首页> 外文期刊>Infection and immunity >Brucella abortus Strain RB51 as a Vector for Heterologous Protein Expression and Induction of Specific Th1 Type Immune Responses
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Brucella abortus Strain RB51 as a Vector for Heterologous Protein Expression and Induction of Specific Th1 Type Immune Responses

机译：流产布鲁氏菌菌株RB51作为异源蛋白表达和诱导特异性Th1型免疫反应的载体

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Brucella abortus strain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, β-galactosidase of Escherichia coli, and the 65-kDa heat shock protein (HSP65) of Mycobacterium bovis in strain RB51. We cloned the promoter sequences of Brucella sodC andgroE genes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for β-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of β-galactosidase expression is higher under the groE promoter than under the sodCpromoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of β-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the β-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with β-galactosidase induced the secretion of gamma interferon (IFN-γ), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-γ, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of β-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or its vaccine efficacy against B. abortus 2308 challenge.
机译：流产布鲁氏菌菌株RB51是一种稳定，粗糙，减毒的突变体，广泛用作牛布鲁氏菌病的活疫苗。我们的最终目标是开发RB51菌株，作为传递其他细胞内病原体保护性抗原的优先载体，有效诱导需要强烈诱导Th1型免疫应答。作为朝这个方向迈出的第一步，我们研究了外源报道蛋白，大肠杆菌β-半乳糖苷酶和牛分枝杆菌在RB51菌株中表达的65 kDa热激蛋白（HSP65）。我们在pBBR1MCS中克隆了布鲁氏菌sodC和groE基因的启动子序列，分别产生质粒pBBSODpro和pBBgroE。将β-半乳糖苷酶（lacZ）和HSP65的基因克隆到这些质粒中，并用于转化菌株RB51。重组RB51菌株中的酶分析表明，在groE启动子下，β-半乳糖苷酶的表达水平高于在sodC启动子下。在含有pBBgroE / lacZ但不包含pBBSODpro / lacZ的RB51菌株中，对细菌进行热激或内化成巨噬细胞样J774A.1细胞后，观察到β-半乳糖苷酶表达水平升高。接种了表达β-半乳糖苷酶的重组RB51菌株的小鼠均产生了主要是免疫球蛋白G2a（IgG2a）同种型的特异性抗体，并在体外用β-半乳糖苷酶刺激了脾细胞诱导了γ干扰素（IFN-γ）的分泌，但不是白介素4（IL-4）。还检测到Th1型对HSP65的免疫反应，这是由特异性抗原刺激的脾细胞分泌的特异性血清IgG2a而非IgG1抗体和IFN-γ而非IL-4的存在所表明的。用含有pBBgroE / hsp65的RB51疫苗接种的小鼠。小鼠研究表明，β-半乳糖苷酶或HSP65的表达不会改变RB51菌株的减毒特性或其对流产双歧杆菌2308攻击的疫苗效力。

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