目的 研究鸟胞内分枝杆菌复合群(MAC)MAV3104基因编码蛋白与药物外排的关系。方法 以MAC标准株基因组为模板,扩增MAV3104基因,构建pMV261-MAV3104c重组质粒;测序正确后,转化重组质粒到大肠杆菌DH5并在MAC标准株中诱导表达, Western Blot鉴定MAV3104表达;按照CLSIM24-A2的操作要求检测MAC标准株对克拉霉素的敏感性及外排泵抑制试验。结果 经基因测序及Western Blot蛋白表达验证重组质粒构建成功;MAV3104过表达能提高鸟分枝杆菌对克拉霉素的MIC,且硫利达嗪能抑制该作用;MAV3104过表达也能提高胞内分枝杆菌对克拉霉素的MIC,但硫利达嗪对其没有抑制效应。结论 MAV3104转运蛋白介导的药物外排在鸟分枝杆菌耐克拉霉素中起重要作用。

Objective To investigate the association of the protein coded by Mycobacterium avium-Mycobacterium intracellulare complex in MAV3104 in drug efflux of Clarithromycin. Methods According to the Mycobacterium avium-Mycobacterium intracellulare complex standard strain, the MAV3104 gene was amplified by PCR and cloned into expression vector pMV261 to generate the recombinant plasmids. After verification by sequence analysis, the recombinant plasmids was transformed into E. coli DH5 andMycoba ctenum avium-Mycobacterium intracellulare complex standard strain. To identify the protein expression by western blotting and investigate the sensitivity of clarithromycin and efflux pump inhibition test in the light of CLSIM24-A2 protocol. Results It was verified by sequence analysis and western blotting that the recombinant plasmids were successfully constructed. Over expression of Mycobacterium avium MAV3104 gene could enhance clarithromycin MIC, which could be inhibited by thioridazine. Over expression of Mycobacterium intracellulare MAV3104 gene also could enhance clarithromycin MIC, but it could not inhibited by thioridazine. Conclusion This study demonstrates that efflux pumps mediated by MAV3104 protein play an important role in Mycobacterium avium resistance to clarithromycin.

目的 评价实时荧光定量PCR分析低氧性肺动脉高压大鼠肺动脉平滑肌基因表达时12个候选内参基因表达的稳定性,获得最适合的内参基因。方法 以低氧性肺动脉高压大鼠肺动脉平滑肌为研究对象,选择文献报道的常用12种内参基因为候选内参基因,利用geNorm和NormFinder程序分析实时荧光定量PCR数据,筛选出最适内参基因。结果 12个候选内参基因在低氧性肺动脉高压大鼠肺动脉平滑肌表达稳定性由强到弱顺序为:TBP>B2M>HPRT1>HMBS>RPL13a>18sRNA>PPIA>ACTB>GUSB>TFRC>GAPDH>PGK1,平均表达稳定度(M值)均<0.5,geNorm和NormFinder评估后推荐使用TBP和B2M一起作为该研究时的内参基因。结论 同时使用TBP和B2M是实时荧光定量PCR分析低氧性肺动脉高压大鼠肺动脉平滑肌基因表达的最适合内参基因,为低氧性肺动脉高压相关基因研究提供最优内参基因。

Objective To compare and select the suitable reference genes in real-time quantitative PCR analysis of rat pulmonary artery smooth muscle cells mRNA expression level of pulmonary hypertension. Methods To choose appropriate reference gene, the expression of twelve commonly use housekeeping genes were examined in rat pulmonary artery smooth muscle cells of hypoxia-induced pulmonary hypertension by using geNorm and NormFinder programs. Results The expression consistency of 12 genes was (from high to low): TBP>B2M>HPRT1>HMBS>RPL13a>18sRNA>PPIA>ACTB>GUSB>TFRC>GAPDH>PGK1. The average expression stability(M) values of them were low than 0.5. TBP and B2M reference genes were recommended to use in the same condition. Conclusion TBP and B2M reference genes were the most suitable combination of the reference genes for real-time quantitative PCR analysis in rat pulmonary artery smooth muscle cells of hypoxia-induced pulmonary hypertension.