结核杆菌Ag85B基因疫苗与结核杆菌Ag85B和细胞因子GM-CSF共表达基因疫苗在小鼠体内的免疫效应比较

目的比较结核杆菌Ag85B基因疫苗与结核杆菌Ag85B和细胞因子GM-CSF共表达基因疫苗在小鼠体内的免疫效应,为研制高效结核杆菌基因疫苗奠定基础。方法将雌性C57BL/6健康小鼠54只,随机分为3组,即结核杆菌Ag85B和细胞因子GM-CSF共表达基因疫苗(pIRES-Ag85B/GM-CSF)组、结核杆菌Ag85B基因疫苗(pIRES-Ag85B)组和空载体(pIRES)组,分别小鼠肌内注射疫苗,检测小鼠体内特异性体液免疫和细胞免疫应答相关指标,同时另取C57BL/6小鼠,随机分成4组,第1、2、3组免疫方法与上述各组免疫方法相对应,第4组每只小鼠背部皮下注射1×106CFU卡介苗(BCG),作为阳性对照,进行动物免疫保护性实验,比较分析两种基因疫苗在小鼠体内的免疫效应。结果结核杆菌Ag85B和细胞因子GM-CSF共表达基因疫苗在小鼠体内的免疫原性和免疫保护性明显强于结核杆菌Ag85B基因疫苗。结论结核杆菌Ag85B和细胞因子GM-CSF共表达基因疫苗能增强结核病基因疫苗的免疫原性和免疫保护性。     

结核分枝杆菌双组分系统MprAB的研究进展

双组分系统(two component system,TCS)是结核分枝杆菌(Mycobacterium Tuberculosis,MTB)感应环境因素刺激,并作出相应反应,以适应环境变化的一种跨膜信号转导系统。MprAB作为MTB目前发现的11个完整的TCS之一,与MTB的持留性及调节应激反应等方面密切相关,本文就MTB MprAB TCS的研究进展进行了综述。     

结核分枝杆菌Pup-蛋白酶体系统与不同毒力结核分枝杆菌致病性的相关性研究

目的通过比较分析不同毒力结核分枝杆菌Pup、Dop、PafA和Mpa基因的表达差异,探讨Pup-蛋白酶体系统与不同毒力结核分枝杆菌致病性的相关性。方法分别提取卡介苗菌株(BCG)、结核分枝杆菌国际标准无毒珠(H37Ra)、结核分枝杆菌国际标准强毒珠(H37Rv)、新疆地区流行的优势强毒结核分枝杆菌临床分离株总RNA,并进行纯度鉴定。运用SYBR GreenⅠ实时荧光定量PCR检测结核分枝杆菌Pup、Dop、PafA和Mpa基因的表达,比较分析不同毒力结核分枝杆菌Pup、Dop、PafA和Mpa基因的表达差异。结果 H37Rv菌株和新疆地区流行的优势强毒结核分枝杆菌临床分离株与BCG菌株相比,Pup基因表达分别上调1.67、2.65倍,Dop基因表达分别上调2.42、3.69倍,PafA基因表达分别上调4.09、7.36倍,Mpa基因表达分别上调2.65、3.91倍,差异有统计学意义(P<0.05);BCG、H37Ra、H37Rv菌株与新疆地区流行的优势强毒结核分枝杆菌临床分离株相比,Pup基因表达分别下调62%、59%、37%倍,Dop基因表达分别下调63%、64%、34%倍,PafA基因表达分别下调87%、86%、44%倍,Mpa基因表达分别下调64%、67%、32%倍,差异有统计学意义(P<0.05)。结论在BCG、H37Ra、H37Rv、新疆地区流行的优势强毒结核分枝杆菌临床分离株Pup、Dop、PafA和Mpa基因的表达有差异性,且与菌株毒力呈正相关。Pup-蛋白酶体系统与不同毒力结核分枝杆菌致病性具有相关性。     

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.     

结核杆菌免疫保护性抗原Ag85A的基因克隆与表达研究

目的结核病疫情再度增高给结核病的防治提出了新的挑战,由于卡介苗对结核病的预防效果极不稳定,发展新型结核病疫苗势在必行,本研究通过对结核杆菌免疫保护性抗原Ag85A进行克隆与表达研究,旨在为结核病新型疫苗研制提供新的靶点.方法根据结核杆菌H37Rv株免疫保护性抗原Ag85A的基因序列自行设计一对寡核苷酸引物,以结核杆菌H37Rv株基因组DNA为模板通过PCR扩增获得具有完整开架读码框(ORF)的Ag85A抗原基因,并将其正向插入真核和原核穿梭表达型载体pBKCMV构建重组质粒,重组质粒转入大肠杆菌后IPTG诱导表达,并对其表达产物进行Western-blotting分析.结果①PCR扩增获得了具有完整开架读码框的Ag85A抗原基因;②构建了Ag85A抗原基因真核和原核穿梭表达型载体;③Ag85A抗原基因在大肠杆菌中实现了稳定表达.结论本研究成功地对结核杆菌免疫保护性抗原Ag85A进行了基因克隆与表达,为进一步研究其在结核病新型疫苗研制中的应用奠定了基础.     

卡介苗菌株Hsp16.3基因打靶载体的构建及鉴定

目的 构建卡介苗菌株Hsp16.3基因打靶载体.方法 体外培养卡片介苗菌株并提取基因组DNA,扩增Hsp16.3基因两侧序列5′-Hsp16.3和3'-Hsp16.3,分别插入到pKO质粒载体预定位点中构建Hsp16.3基因置换型打靶载体,筛选并进行PCR、双酶切鉴定及测序鉴定.结果 构建的卡介苗菌株Hsp16.3基因打靶载体,经PCR、双酶切鉴定及测序证实pKO质粒中的2个插入片段为所需目的基因.结论 成功构建出卡介苗菌株Hsp 16.3基因打靶载体.     

应用抑制性消减杂交技术筛选结核分枝杆菌差异基因

目的 筛选新疆地区结核分枝杆菌临床分离株与国际标准株H37Rv之间的差异基因,初步分析这些基因的功能.方法 利用抑制性消减杂交技术,通过两轮两相杂交,驱赶相同基因富集差异基因.结果 正相抑制性消减杂交一共获得6条新疆地区临床分离株中存在的差异基因,虽然这些基因在H37Rv基因组中不存在,但与国际标准临床株CDC1551以及复合群国际标准株KMS中的对应基因高度同源,分别与编码多萜醇磷酸甘露糖基转移酶Ⅱ、单加氧酶黄素结合蛋白、酰基转移酶蛋白、染色体复制起始密码子、脂酰基载体蛋白以及5'-磷酸吡哆胺氧化酶的基因有关.结论 新疆地区结核分枝杆菌临床分离株与国际标准株H37Rv之间存在差异基因,这些差异基因的功能可能与已知同源基因的功能相似:增强细菌胞壁蛋白的吸附能力、抵抗氮氧合酶消化、提高乙酰基辅酶A的合成能力、调控复制起始密码子、合成胞壁脂酰基载脂蛋白和促进5'-磷酸吡哆胺氧化酶代谢.     

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.     

PCR方法鉴定结核分枝杆菌临床分离株

目的:用3对引物PCR扩增的方法对结核分枝杆菌临床分离株进行菌种鉴定.方法:将3对特征性的结核分枝杆菌标记基因:MPT40基因、α抗原基因和IS6100插入序列,分别放入PCR体系中,在各自的退火温度下进行PCR扩增,通过对扩增产物的不同带型进行分析,对结核分枝杆菌临床分离株进行菌种鉴定.结果:用3对引物PCR扩增的方法可将各种类型的结核分枝杆菌以及结核分枝杆菌与非结核分枝杆菌鉴别开.结论:3对引物PCR扩增的方法是一种有效的结核分枝杆菌菌种鉴定的方法,可用于结核分枝杆菌与非结核分枝杆菌以及各种类型的结核分枝杆菌的区分.