结核杆菌H37Rv异柠檬酸裂解酶基因的克隆及表达

目的构建在大肠杆菌中高效表达结核杆菌H37Rv异柠檬酸裂解酶(ICL)的重组质粒,实现ICL在原核表达系统的高效稳定表达.方法采用PCR和克隆技术构建含有结核杆菌H37Rv ICL基因的表达质粒pET30(a)-Rv0467,转化至E.coli BL21(DE3)中诱导表达.目的蛋白经金属螯合层析纯化后,对其活性进行初步研究.结果构建了高效表达结核杆菌H37Rv ICL的质粒;在E.coli BL21(DE3)中得到高效表达,目的蛋白占总蛋白含量的30%;表达产物以可溶性形式存在,通过金属螯合层析纯化,所得酶的纯度为90%;目的蛋白具有ICL的活性.结论利用原核表达系统成功克隆表达了结核杆菌H37Rv的ICL,为以ICL为靶点建立新型抗结核药物奠定了基础.     

结核分枝杆菌RD1区Rv3873基因真核表达载体的构建及鉴定

[目的]构建结核分枝杆菌RD1区Rv3873基因的真核表达载体pcDNA3-Rv3873，并对其进行鉴定。[方法]利用PCR技术从结核杆菌H37Rv中扩增Rv3873基因，并将其定向克隆至原核载体pGEx-4T-1中，经测序鉴定后，将重组质粒pGEX-4T-1-Rv3873中的目的基因亚克隆入真核表达载体pcDNA3．1（＋），构建重组子pcDNA3．1-Rv3873，通过限制性内切酶切酶及PCR进行鉴定。[结果]克隆的Rv3873基因序列与GenBank公布的一致性为100％，限制性内切酶酶切及PCR分析表明Rv3873已成功插入pcDNA3．1（＋）中。[结论]成功构建了Rv3873基因真核表达载体，为进—步研究新型结核杆菌DNA疫苗奠定了基础。     

结核杆菌重组Rv3425蛋白的结核病血清学诊断价值的评价

[目的]评价结核分支杆菌重组Rv3425蛋白在结核病血清学诊断中的价值。[方法]以重组Rv3425作为抗原，采用酶联免疫吸附试验（ELISA）检测151份确诊结核病人、143份健康人、38份卡介苗接种阳转者和42份非结核病人血清中的抗结核抗体水平，并与结核菌素纯蛋白衍生物（PPD）、Ag85B蛋白及TB—DOT、TB-CHECK-1试剂盒的检测结果进行比较，以此来评价重组Rv3425蛋白在结核病诊断上的价值。[结果]Rv3425与PPD和Ag85B抗原一样能够诊断结核病人，但PPD不能有效区分结核病人和BCG接种者，Rv3425和Ag85B蛋白可以明确区分。[结论]虽然重组Rv3425蛋白对结核病的阳性率尚不能与试剂盒媲美，但由于其能够有效区分结核病人和BCG阳转者，在结核病诊断方面具有很大的应用价值，可以作为结核病诊断的备选抗原之一。     

结核病生物诊断试剂豚鼠模型建立

目的建立结核病生物诊断试剂模型。方法通过在豚鼠腹股沟皮下单次和多次注射不同剂量灭活H37Rv全菌体,制备结核病生物诊断试剂模型,采用0.1 mL(5 IU)标准结核菌素(TB-PPD)对模型效果进行评价。结果三组剂量(0.2 mg/mL,0.3 mg/mL和0.5 mg/mL)致敏豚鼠均能建立模型,其中0.2 mg/mL剂量组皮试后24 h和48 h红斑纵横径值均大于0.3 mg/mL和0.5 mg/mL剂量组,致敏效果最好;多次致敏皮试红斑纵横径值与单次致敏比较无显著差异;第1次皮试后24 h红斑纵横径值大于第2次、第3次和第4次皮试后红斑纵横径值(P≤0.05);此外,灭活H37Rv全菌体致敏后,豚鼠体重持续增加,部分豚鼠注射部位有溃烂发生。结论应用0.2mg/mL灭活H37Rv全菌体单次致敏豚鼠,可以建立良好的用于结核病生物诊断试剂评价的模型。增加致敏剂量和致敏次数不能增加变态反应强度。     

结核杆菌抗原Rv0173的HLA-A*0201限制性CTL表位的预测及鉴定

目的 鉴定结核杆菌(Mycobacterium tuberculosis,Mtb)抗原Rv0173中的HLA-A*0201限制性CTL表位.方法 应用数据库SYFPETTHI预测结核杆菌Rv0173中可能存在的HLA-A*0201限制性CTL表位,经流式细胞术分析各抗原肽与HLA-A*0201的亲合力,经时间分辨荧光法检测外周血单个核细胞(PBMCs)对各抗原肽产生的增殖反应,经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性,逐步鉴定Rv0173的HLA-A*0201限制性CTL表位.结果 位于Rv0173氨基酸序列肽3(21-30aa)和抗原肽7(161-170aa)与HLA-A*0201分子具有较高的亲合力,并都能刺激HLA-A*0201阳性个体PBMCs增殖,并诱导产生特异性杀伤活性.结论 肽3(RLSQSADQYL)(21-30aa)和肽7(LLRGGGLVNL)(161-170aa)是抗原Rv0173上HLA-A*0201限制性CTL的优势表位,可作为结核疫苗设计的候选表位.     

Associations Between Insurance Provider and Assistive Technology Use for Computer and Electronic Devices 1 Year After Tetraplegia: Findings From the Spinal Cord Injury Model Systems National Database

ObjectiveTo investigate the association between insurance provider and reported assistive technology (AT) use to access computers and electronic devices 1 year after sustaining tetraplegia.DesignMulticenter cross-sectional study.SettingParticipants enrolled in the Spinal Cord Injury Model Systems (SCIMS) National Database.InterventionsNot applicable.ParticipantsMen and women with tetraplegia (N=498) enrolled in the SCIMS National Database were included in the analysis.Main Outcome MeasuresThe primary study outcome was the use of AT when operating a computer or other mobile electronic device. The primary predictor was the subject’s principal health insurance provider, which was grouped into the 3 categories: government (Medicare, Medicaid, and other government), private (private insurance, private funds, and other), and workers’ compensation.ResultsOverall, 34.7% of participants reported using AT to access computers and electronic devices. Results of logistic regression analysis revealed sex, injury level, injury completeness, self-perceived health status, and 12-month history of pressure ulcer were all significantly associated with AT use. After adjusting for these factors, participants with workers’ compensation were more likely to report AT use than individuals with either government or private insurance.ConclusionsDespite significant technological advances, AT is not readily available to the people who might benefit most from its use. Findings from the present study are the first to shed light on AT funding sources and reveal that individuals with workers’ compensation are more likely use AT than individuals with either government or private insurance. Additional work focused on AT use and functional outcomes is needed to assess the effect of barriers to use. Collectively, this work may inform insurers of the importance of having AT available for this unique population to potentially improve quality of life and participation.     

耐利福定结核杆菌膜屏障通透性改变的研究

选用结核杆菌H_(37)Rv及其体外诱导耐利福定菌株,发现利福定能明显抑制~3H-尿苷掺入敏感菌,而~3H-尿苷掺入耐药菌则不受抑制,~3H-利福定掺入敏感菌的量明显高于耐药菌的量;透射电镜观察发现耐药菌包膜较敏感菌为厚。提示利福定抗结核杆菌机理与利福平相同,也是抑制RNA合成;结核杆菌耐利福定与其菌膜屏障通透性降低有关。