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牛种布鲁氏菌A19ΔBtpA缺失株生物学特性及其免疫原性研究 被引量:1

Biological Characteristics of Brucella abortus A19ΔBtpA Deletion Strainand Its Immunogenicity Study
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摘要 布鲁氏菌四型分泌系统(T4SS)效应物BtpA是布鲁氏菌的重要免疫抑制分子之一。为了进一步了解BtpA对牛种布鲁氏菌生物学特性的影响,并探究BtpA基因缺失是否会影响布鲁氏菌的免疫原性。在牛种布鲁氏菌疫苗株(A19株)上对BtpA基因进行了缺失,通过qRT-PCR检测布鲁氏菌A19ΔBtpA缺失株中的BtpA mRNA表达水平,并探究布鲁氏菌A19株和A19ΔBtpA缺失株在生长曲线、胞内生存、黏附侵袭和体外应激方面的差异。此外,将A19株和A19ΔBtpA缺失株免疫小鼠,在免疫后的第7、14、21、28和35天使用间接ELISA检测小鼠体内的布鲁氏菌特异性抗体水平;通过ELISpot检测免疫第21天时小鼠脾淋巴细胞中IFN-γ的表达水平,利用流式细胞术测定了小鼠脾淋巴细胞中IFN-γ阳性CD4^(+)、CD8^(+)T淋巴细胞以及CD4^(+)、CD8^(+)T淋巴细胞的分类情况。结果表明:成功构建了A19ΔBtpA缺失株,A19ΔBtpA缺失株中BtpA的转录水平显著低于A19株,A19ΔBtpA缺失株与亲本株在生长曲线、胞内生存和黏附侵袭方面呈现相似的趋势。而体外应激试验显示,高渗应激条件下A19ΔBtpA缺失株的存活菌量明显低于A19株(P<0.05)。免疫原性研究中,与PBS组相比,A19组和A19ΔBtpA组均可极显著诱导小鼠产生布鲁氏菌特异性抗体(P<0.01);与A19组相比,A19ΔBtpA组淋巴细胞IFN-γ表达水平显著升高(P<0.05),使小鼠产生的IFN-γ阳性CD4^(+)、CD8^(+)T淋巴细胞以及CD4^(+)/CD8^(+)T细胞比值显著增高(P<0.05)。BtpA基因缺失不会影响布鲁氏菌A19体外胞内增殖,但可能参与布鲁氏菌抗高渗环境能力相关。此外,A19ΔBtpA缺失株比A19株更好地诱导Th1型免疫应答,诱导宿主产生与A19株相当的IgG特异性抗体,具有布鲁氏菌基因缺失疫苗的潜力。该研究将为进一步探究布鲁氏菌的致病机制和开发基因缺失株疫苗提供理论基础和数据支持。 The Brucella type IV secretion system(T4SS)effector BtpA plays a significant role as an immunosuppressive molecule in Brucella.This study aims to gain a deeper understanding of the impact of BtpA on the biological properties of Brucella abortus strain A19 and to explore the potential effect of BtpA gene deletion on the immunogenicity of strain A19.The BtpA gene was deleted on a bovine Brucella vaccine strain(strain A19).qRT-PCR was performed to detect the BtpA mRNA expression level in the Brucella A19ΔBtpA deletion strain and to investigate the differences between Brucella A19 and A19ΔBtpA deletion strains with respect to growth curves,intracellular survival,adherence invasion and in vitro stress.In addition,mice were immunized with the A19 and A19ΔBtpA deletion strains,and Brucella-specific antibody levels were measured using iELISA at the 7th,14th,21st,28th and 35th day post-immunization;the expression level of IFN-γin mouse splenic lymphocytes at day 21 of immunization was detected by ELISpot.IFN-γ-positive CD4^(+)and CD8^(+)T lymphocytes and the classification of CD4^(+)and CD8^(+)T lymphocytes were determined by flow cytometry.The A19ΔBtpA deletion strain was successfully constructed,and the transcript level of BtpA in the A19ΔBtpA deletion strain was significantly lower than that of the A19 strain,and the A19ΔBtpA deletion strain and the parental strain showed a similar trend in terms of the growth curve,intracellular survival,and adhesion invasion.In contrast,the in vitro stress test showed that the surviving bacterial population of the A19ΔBtpA deletion strain was significantly lower than that of the A19 strain under hypertonic stress conditions(P<0.05).In the immunogenicity study,both A19 and A19ΔBtpA groups induced the production of Brucella-specific antibodies in mice very significantly compared with the PBS group(P<0.01);the IFN-γexpression level of lymphocytes in the A19ΔBtpA group was significantly higher compared with that of the A19 group(P<0.05),which resulted in the production of IFN
作者 徐朕宇 邓肖玉 王月丽 孙灿 吴澳迪 曹剑 易继海 王勇 王震 陈创夫 XU Zhenyu;DENG Xiaoyu;WANG Yueli;SUN Can;WU Aodi;CAO Jian;YI Jihai;WANG Yong;WANG Zhen;CHEN Chuangfu(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;Collaborative Innovation Center for Healthy Breeding and Prevention and Control of Human-Veterinary Diseases in Sheep,Shihezi 832000,China;Tiankang Biopharmaceutical Co.,Ltd.,Urumchi 830011,China;Key Laboratory of Animal Disease Prevention and Control Corps,Shihezi University,Shihezi 832000,China)
出处 《畜牧兽医学报》 CAS CSCD 北大核心 2024年第5期2135-2145,共11页 ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金 国家自然科学基金项目(32002245,U1803236)。
关键词 布鲁氏菌 BtpA 生物学特性 免疫原性 Brucella BtpA biological properties immunogenicity
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