应用抑制性消减杂交技术筛选结核分枝杆菌差异基因

Objective To screen differential Mycobacterium tuberculosis genes between Xinjiang clinical strains and H37Rv by suppression subtractive hybridization( SSH), and to analyze the function of these specifically pathopoiesis genes. Methods Both M. tuberculosis Xinjiang clinical strains and H37Rv as tester and driver each other, most identical genome was drived whereas some distinctive genes was re-mained and enriched by utilization SSH technique. Meanwhile through inserting differential genes to E. coli all of sequences that we have cloned were determined by BLAST in GenBank. The function of differential genes between M. tuberculosis H37Rv and Xinjiang clinical strains were analyzed. Results We cloned and analyzed six different DNA fragments that only existed in Xinjiang clinical strains. One is the fragment of a gene ceding monooxygenase, flavin-binding family identified by Glimmer2. One fragment belongs to acyl-transferase family protein. One for aminotransferase, class Ⅱ, acyl carrier protein. One fragment belongs to chromosomal replication initiator protein DNA and one for M. tuberculosis paralogous family 11-pyridoxamine 5'-phosphate oxidase-related. Meanwhile, we cloned ten DNA fragments only in H37Rv. Conclusion SSH technique can efficiently screen differential genes in M. tuberculosis in Xinjiang clinical strains. They are possible key genes that M. tuberculosis survive and fortify virulence in mal-environment as same as their ho-mogenic genes, such as enhanced adsorbability in wall-held protein, counteracted digestion by nitro-oxygen-ase, elevated composition capability in the acyhransferase, control chromosomal replication initiator protein, synthesized aminotransferase acyl cartier protein and pyridoxamine 5'-phosphate oxidase.     

应用抑制性消减杂交技术筛选结核分枝杆菌差异基因

Objective To screen differential Mycobacterium tuberculosis genes between Xinjiang clinical strains and H37Rv by suppression subtractive hybridization( SSH), and to analyze the function of these specifically pathopoiesis genes. Methods Both M. tuberculosis Xinjiang clinical strains and H37Rv as tester and driver each other, most identical genome was drived whereas some distinctive genes was re-mained and enriched by utilization SSH technique. Meanwhile through inserting differential genes to E. coli all of sequences that we have cloned were determined by BLAST in GenBank. The function of differential genes between M. tuberculosis H37Rv and Xinjiang clinical strains were analyzed. Results We cloned and analyzed six different DNA fragments that only existed in Xinjiang clinical strains. One is the fragment of a gene ceding monooxygenase, flavin-binding family identified by Glimmer2. One fragment belongs to acyl-transferase family protein. One for aminotransferase, class Ⅱ, acyl carrier protein. One fragment belongs to chromosomal replication initiator protein DNA and one for M. tuberculosis paralogous family 11-pyridoxamine 5'-phosphate oxidase-related. Meanwhile, we cloned ten DNA fragments only in H37Rv. Conclusion SSH technique can efficiently screen differential genes in M. tuberculosis in Xinjiang clinical strains. They are possible key genes that M. tuberculosis survive and fortify virulence in mal-environment as same as their ho-mogenic genes, such as enhanced adsorbability in wall-held protein, counteracted digestion by nitro-oxygen-ase, elevated composition capability in the acyhransferase, control chromosomal replication initiator protein, synthesized aminotransferase acyl cartier protein and pyridoxamine 5'-phosphate oxidase.     

利用基因芯片技术快速检测结核杆菌耐药性的初步研究

目的：探讨利用基因芯片技术检测结核杆菌的耐药性。方法：结核杆菌耐药性检测基因芯片的制备；临床分离株结核杆菌培养和药敏检测；结核杆菌基因组DNA的制备和结核杆菌耐药基因的聚和酶链反应；基因芯片的杂交、洗涤、检测与分析。结果：临床分离株结核杆菌培养和药敏检测结果：1株为药物敏感株结核杆菌(对异烟肼、利福平、链霉素、乙肤丁醇4种药物均敏感)；4株为多耐药株结核杆菌(对异烟肼、利福平、链霉素、乙肤丁醇4种药物均耐药)；药敏检测结果与5位患者临床的诊断性治疗的结果完全一致；进一步利用基因芯片技术进行检测，检测结果与上述结果相符合。结论：利用基因芯片技术检测结核杆菌的耐药性，准确、快速、高效。     

新型结核病疫苗菌株对T淋巴细胞免疫记忆及增殖能力的影响研究

背景　结核病是世界范围内单一致病菌感染导致死亡率最高的慢性感染性疾病,目前缺乏安全有效的疫苗预防成人结核病。而预防接种主要是利用机体具有免疫记忆的特征以阻止病原体的再次入侵,因此,研究T淋巴细胞的免疫记忆及增殖能力对新型结核病疫苗的设计至关重要。目的　探讨新型结核病疫苗菌株（简称B/R菌株）对T淋巴细胞免疫记忆及增殖能力的影响。方法　2019年1月,选取6~8周龄、SPF级雌性C57BL/6小鼠48只,随机分为磷酸盐缓冲液（PBS）组、卡介苗（BCG）组、结核分枝杆菌国际标准毒株（H37Ra）组和新型结核病疫苗（B/R）菌株组,各12只。PBS组、BCG组、H37Ra组和B/R菌株组分别皮下接种PBS、BCG、H37Ra和B/R菌株。4组分别于免疫后8周、12周、16周各取3只小鼠提取脾淋巴细胞体外刺激培养,利用流式细胞技术检测中央型记忆性淋巴细胞（TCM）及效应型记忆性淋巴细胞（TEM）,酶联免疫吸附试验（ELISA）检测脾淋巴细胞分泌的抗原特异性Th1/Th2型细胞因子〔白介素2（IL-2）、干扰素γ（IFN-γ）和白介素4（IL-4）〕水平;羟基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记法检测CD4+T淋巴细胞增殖率、CD8+ T淋巴细胞增殖率。结果　免疫后8、12、16周BCG组、H37Ra组、B/R菌株组CD4+的TCM和TEM高于PBS组（P<0.05）;免疫后8周B/R菌株组CD4+的TEM低于H37Ra组（P<0.05）;免疫后16周B/R菌株组CD4+的TCM高于H37Ra组和BCG组（P<0.05）;免疫后16周B/R菌株组CD4+的TEM高于BCG组（P<0.05）。免疫后8、12、16周BCG组、H37Ra组、B/R菌株组CD8+的TCM和TEM高于PBS对照组（P<0.05）;免疫后12周B/R菌株组CD8+的TEM高于H37Ra组和BCG组（P<0.05）;免疫后16周,B/R菌株组CD8+的TCM和TEM高于BCG组（P<0.05）。免疫后8、12、16周PBS组IL-2和IFN-γ低于BCG组、H37Ra组、B/R菌株组（P<0.05）;免疫后12周,B/R菌株组IFN-γ高于BCG组（P<0.05）;免疫后16周,B/R菌株组IFN-γ和IL-2高于BCG组和H37Ra组（P<0.05）。BCG组、H37Ra组和B/R菌株组CD4+T淋巴细胞增殖率、CD8+ T淋巴细胞增殖率高于PBS组（P<0.05）;B/R菌株组的CD4+T淋巴细胞增殖率、CD8+T淋巴细胞增殖率均高于BCG组和H37Ra组（P<0.05）。结论　B/R菌株免疫小鼠后,可促进机体产生更高水平的免疫保护性记忆细胞,以Th1型细胞免疫应答为主,并可增强T淋巴细胞增殖能力。     

结核分枝杆菌PhoP蛋白的生物信息学分析

目的应用生物信息学软件预测结核分枝杆菌PhoP基因编码蛋白的结构和功能。方法从NCBI数据库获取PhoP基本的基因信息及其编码序列氨基酸信息;应用ProtParam软件预测PhoP蛋白的理化性质;利用SignalIP4.1和TMHMM分析其信号肽和跨膜区;利用在线分析Expasy工具分析蛋白二级结构,建立蛋白三级结构模型;采用Bepipred 1.0Server和SYFPEITHI分析蛋白的B细胞及T细胞抗原表位。结果 PhopP蛋白共250个氨基酸,分子式为C1226H1974N346O364S4,原子总数3 914,半衰期为30h,预测该蛋白为稳定性亲水性蛋白;二级结构中α-螺旋、β-转角、β-折叠、无规则卷曲分别占36.03%、10.58%、24.7%和27.94%,预测的B细胞、CTL细胞抗原表位分别为21个和7个;PhoP基因与天蓝色链霉菌同源性较高;其编码蛋白的相互作用蛋白为PhoR、senX3、trcS等。结论生物信息学分析PhoP为稳定蛋白,且含有潜在的B、T细胞抗原表位,是结核病诊断及治疗的潜在候选因子。     

维生素D受体基因多态性与新疆维吾尔族人群结核病易感性的相关性研究

目的研究维生素D受体(VDR)基因多态性与新疆维吾尔族人群结核病易感性的相关性。方法采用病例对照研究,选取224例新疆维吾尔族结核患者和225例有分枝杆菌感染史的同民族健康者分别作为病例组和对照组。用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)分析方法分别检测维生素D受体基因的多态性,内切酶分别为FokI和TaqI,并进行基因分型。统计学分析结果采用χ2检验,P值为0.05。结果FokI分析,分为FF、Ff、ff三种基因型,其在病例组和对照组中分别为36%、57%、7%和40%、54%、6%。FF基因型的分布在病例组与对照组中的差异性无统计学意义(χ2=0.872,P>0.05)。TaqI分析,分为TT、Tt、tt三种基因型,其在病例组和对照组中分别为68.8%、29.4%、1.8%和75.6%、22.2%、2.2%。TT基因型在病例组与对照组中的差异性也无统计学意义(χ2=2.588,P>0.05)。结论维生素D受体基因中FokI与TaqI多态性与新疆维吾尔族人群结核病易感性无明显相关性。     

卡介苗ERP基因置换型打靶载体的构建

目的：构建ERP基因打靶载体。方法：卡介前菌体外培养,扩增ERP基因两侧序列．连接载体与目的片段．筛选阳性克降并鉴定。结果：PCR扩增捕入片段大小与预期相符．鉴定证实PCR产物及插入片段为所需目的基因片段。结论：成功构建了用于卡介苗菌基因打靶的置换型载体,为今后构建ERP基因敲除株的卡介苗突变株的研究奠定基础。     

结核病疫苗研究进展

本文从亚单位疫苗、DNA疫苗、重组卡介苗和转基因植物疫苗等方面概括介绍了目前新型结核病疫苗研制的现状.     

自体吞噬在机体抗结核分支杆菌感染中的作用

结核分支杆菌长期处于持续感染状态是消灭结核病的最大阻碍之一。阻断吞噬体和溶酶体融合这一成熟过程是结核分支杆菌持续感染的最主要因素之一。近年来研究发现,诱导自体吞噬可以促进吞噬溶酶体的成熟,进而杀灭胞内寄生的结核分支杆菌。现就结核分支杆菌阻断吞噬体一溶酶体融合的分子机制、自体吞噬的分子机制,以及自体吞噬在促进结核分支杆菌吞噬体与溶酶体融合中的作用等作一综述。     

探讨慢性阻塞性肺疾病的第1秒用力呼气容积与第6秒用力呼气容积比值的诊断价值

慢性阻塞性肺疾病(COPD)是一种具有气流受限特征的肺部疾病,气流受限不完全可逆,其气流受限多通过第1秒用力呼气容积(FEV1.0)和FEV1.0与用力肺活量(FVC)的比例(FEV1.0/FVC)减少来确定,其中FEV1.0/FVC是一项敏感指标,但FEV1.0/FVC有时可因技术等原因出现假阳性或假阴性.近年的研究表现,用FEV1.0与第6秒用力呼气容积(FEV6.0)的比例(FEV1.0/FEV6.0)代替FEV1.0/FVC,可减少门诊检查的误诊率,但两者在判断COPD的气流阻塞中,一致性如何,有何差异,国内尚未见到文献报告,我们对其进行了前瞻性研究,报告如下.