Subjects: Biology >> Bioengineering submitted time 2019-03-05 Cooperative journals: 《中国生物工程杂志》
Abstract： Objective To explore the role and mechanism of miR-17-5p in the autophagy pathway mediated by Mycobacterium tuberculosis by studying the regulatory mechanism of miR-17-5p on autophagy-related gene ATG7 and its effect on cell autophagy. Methods The target gene ATG7 of miR-17-5p was obtained by bioinformatics analysis. The wild-type(pMirGLO-ATG7-3'UTR-WT) and mutant vector of ATG7 were successfully constructed. The targeting relationship between miR-17-5p and ATG7 was verified by double luciferase reporting system and Western blot. THP-1-derived macrophages Infected by Mycobacterium tuberculosis (H37Ra) were divided into three groups: miR-17-5p mimics, miR-17-5p inhibitors, and miR-17-5p nc. The effect of H37Ra infection on the expression of miR-17-5p was detected by quantitative real-time PCR (qRT-PCR). The expression of LC3 protein and the number of autophagosomes were detected by Western blot and immunofluorescence. Results MTB infection can cause miR17-5p down-regulation, with the increase of infection plural decreased significantly. Bioinformatics predictions showed that miR-17-5p and ATG7 were targeted. Dual luciferase reporter assay and Western blot confirmed that miR-17-5p could bind to ATG7 and negatively regulate it. Western blot and immunofluorescence assay showed that the expression of LC3 II was down-regulated and the expression of autophagosomes was down-regulated in the miR-17-5p mimics group, but the reverse was found in the miR-17-5p inhibitor group. The expression of ATG7 and LC3 II protein in H37Ra infected group was higher than that in uninfected group. Conclusion MiR-17-5p directly targets ATG7 3'UTR to inhibit autophagy and plays a role in the anti-MTB effect of macrophages.
Subjects: Biology >> Bioengineering submitted time 2018-12-25 Cooperative journals: 《中国生物工程杂志》
Abstract：目的 构建能够用于稳定动态监测细胞自噬流变化和过表达基因的红色荧光蛋白-绿色荧光蛋白-鼠源LC3融合慢病毒多功能表达载体(PCDH-Duo-mRFP-eGFPph-LC3rat，PCDH-Duo) , 并构建小鼠腹腔巨噬细胞Raw264.7稳转株观察自噬流变化。方法 应用基于PCR精确合成mRFP-eGFPph-LC3rat融合全基因，将其克隆至慢病毒表达载体PCDH-CMV-MCS-EF1a-GFP中，重组质粒经菌落PCR、酶切及测序分析正确无误后, 包装慢病毒，转染Raw264.7细胞，并利用流式分选术获取稳转株，经氯喹抑制自噬模型及Western blotting鉴定eGFP蛋白表达确认其可靠性。结果 成功构建了PCDH-Duo重组慢病毒质粒，包被慢病毒并获得Raw264.7稳定细胞系(Raw264.7-PCDH-Duo)，可稳定表达双荧光蛋白，经3 mM氯喹作用6h后，能够稳定准确指示自噬流变化。结论 成功构建了基于慢病毒系统的双荧光标记多功能自噬流监测系统，为研究细胞自噬与编码基因及非编码基因之间的关系提供了方便有力的工具。
Subjects: Biology >> Bioengineering submitted time 2018-10-09 Cooperative journals: 《中国生物工程杂志》
Abstract：目的：构建噬菌体天然纳米抗体展示库，以期用于筛选不同抗原分子的纳米抗体筛选平台，并用艰难梭菌谷氨酸脱氢酶（GDH）抗原筛选靶向GDH的纳米抗体，对所构建的噬菌体天然纳米抗体展示库进行验证。 方法：采用Oligo DT提取双峰骆驼脾脏总RNA进行反转录，通过巢氏PCR获取全套重链可变区基因，将其构建到噬菌粒pCANTAB5E载体，经多次电转化至E.coil TG1 构建初级噬菌体抗体库，经辅助噬菌体拯救后构成噬菌体展示库，并对噬菌体展示库的库容及多样性进行分析和鉴定。同时以GDH为靶向抗原对文库进行淘筛，计算淘筛回收率，并对第三轮淘筛后平板的单克隆进行ELISA鉴定。 结果：构建的天然噬菌体纳米抗体库的插入率为95%左右，随机挑取的9个克隆氨基酸同源性为66.17%，经MEGA分析后具有较好的多样性，同时经辅助噬菌体拯救后，得到的噬菌体展示库滴度为4×1012 CFU/ml。在三轮淘筛过程中，回收率逐步升高，噬菌体得到了有效的富集，同时对阳性克隆进行测序及分析，最终得到2条抗GDH纳米抗体序列。结论：成功构建了双峰驼源天然噬菌体纳米抗体展示文库且多样性良好，为后续筛选其他的靶向抗原奠定了基础，同时筛选获得两条抗GDH纳米抗体序列，为制备艰难梭菌谷氨酸脱氢酶诊断抗体提供技术支撑。
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