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自噬相关5蛋白对刚地弓形虫刺激树突状细胞成熟的影响

Effects of recombinant autophagy related 5 protein on the maturation of dendritic cells stimulated by Toxoplasma gondii
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摘要 目的探讨自噬相关5 (ATG5)蛋白对刚地弓形虫(简称弓形虫)体外刺激树突状细胞(DCs)成熟的影响。方法采用TRIzol法提取昆明小鼠总RNA,反转录为cDNA,经PCR扩增获得ATG5基因。构建重组质粒pET-32a-ATG5,转入大肠埃希菌Rosetta株中表达,尿素纯化后用抗His标签单抗蛋白质印迹(Western blotting)鉴定ATG5。分离鼠源DCs,用弓形虫体外感染DCs。感染实验分为3组:对照组(仅含DCs)、弓形虫组(弓形虫+DCs)和ATG5蛋白组(ATG5蛋白+弓形虫+DCs),每组设3次重复,各组弓形虫速殖子(1×10^4个)同时加入到DCs中, 37℃、 5%CO2共培养2 h。其中, ATG5蛋白组弓形虫在感染DCs前,预先与ATG5蛋白(50μg)在体外作用2 h。采用流式细胞术检测CD11cCD83双表达成熟型DCs在细胞总数中比例的变化,分析DCs的成熟情况,评价ATG5蛋白对弓形虫体外刺激DCs成熟的影响。结果ATG5扩增产物约为729 bp,ATG5蛋白相对分子质量(Mr)约为46 000。Western blotting检测结果显示,重组ATG5蛋白具有良好的反应原性。流式细胞术结果显示,对照组、弓形虫组、 ATG5蛋白组中CD11cCD83双表达成熟型DCs在细胞总数的比例分别为(13.3±0.5)%、(52.5±0.3)%和(47.8±0.6)%,弓形虫组和ATG5蛋白组差异有统计学意义(P 〈0.01),对照组与其他两组的差异有统计学意义(P 〈0.01)。结论ATG5蛋白可降低弓形虫体外刺激DCs成熟的能力。 Objective To investigate the effect of autophagy related 5(ATG5)proteinon the maturation of dendritic cells(DCs)stimulated by Toxoplasma gondii.Methods Total RNA was extracted from Kunming mice using TRIzol,reverse transcribed into cDNA,and amplified by PCR to receive ATG5 gene.The recombinant plasmid pET-32a-ATG5 was constructed and transformed into Escherichia coli Rosetta strain.The expressed recombinant ATG5 protein was purified by urea and identified by Western blotting with anti-His tag mAb.Mouse DCs were isolated,and then infected with T.gondii,which were treated by recombinant ATG5 protein in vitro.There were three groups:control group,T.gondii group(T.gondii+DCs),and ATG5 protein group(ATG5+T.gondii+DCs),each with 3 replicates.The T.gondii tachyzoites(1×10^4)were added to the DCs and incubated at 37℃under 5%CO2 for 2 h.The T.gondii in the ATG5 protein group were pre-treated with ATG5 protein(50μg)in vitro for 2h.The percentage of CD11cCD83 mature DCs among all DCs was assessed by flow cytometry.The effect of ATG5 protein on the maturation of DCs stimulated by T.gondii was evaluated.Result The amplified product of ATG5 was 729 bp,and the relative molecular mass(Mr)of the recombinant ATG5 protein was 46 000.Western blotting showed that the expressed recombinant ATG5 protein had good reactivity.Results of flow cytometry showed that the percentages of CD11cCD83 mature DCs in the control group,T.gondii group,and ATG5 protein group were(13.3±0.5)%,(52.5±0.3)%and(47.8±0.6)%,respectively,with a significant difference between the latter two groups(P〈0.01),and between the control group and the other two(P〈0.01).Conclusion The ATG5 protein can reduce the ability of T.gondii to stimulate the maturation of DCs in vitro.
作者 吴尚桦 耿娥燕 张静 张衡 施之强 汪汕 陆维 吴以振 王桂军 王勇 徐前明 WU Shang-hua, GENG E-yan, ZHANG Jing, ZHANG Heng, SHI Zhi-qiang, WANG Shan, LU Wei, WU Yi-zhen, WANG Gui-jun, WANG Yong, XU Qian-ming (Anhui Agriculture University, Hefei 230036, China)
机构地区 安徽农业大学
出处 《中国寄生虫学与寄生虫病杂志》 CSCD 北大核心 2018年第5期464-468,共5页 Chinese Journal of Parasitology and Parasitic Diseases
基金 安徽省自然科学基金(No.KJ2016a838) 安徽省大学生创新基金(No.201510364017)
关键词 刚地弓形虫 自噬相关5蛋白 树突状细胞 Toxoplasma gondii Autophagy-related 5 protein Dendritic cells
作者简介 通讯作者,E-mail:xuqianming2006@163.com
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