Fast and Accurate Identification of <i>M. tuberculosis</i>Complex Using an Immunochromatographic MPT64 Antigen Detection Test

Background: A new rapid Immunochromatographic test (ICT) kit (MPT64 TB Ag Kit) for detection of MPT64 Antigen in M. tuberculosis (MTB) isolates used for rapid identification of MTB isolates developed by SD (Standard Diagnostics) Bio line, South Korea was evaluated. The ICT is a rapid, reliable and cheaper method that can be used instead of conventional biochemical tests for confirming MTB in culture isolates in resource limited laboratories. The study also evaluated the ability of ICT to detect MPT64-Antigen before the micro MGIT could signal positive. Material/Methods: A total of 450 sputum samples of individual patients were used for the study. 152 isolates of Mycobacteria were recovered from solid and liquid media. These strains were tested for the detection of MPT64-antigen. H37Rv strain was served as the positive reference control and also used for early detection of Antigen experiment. Findings: The development of bands on both test and sample region when H37Rv strain was tested were seen (MPT64 antigen positive). When 138 MTB isolates were tested, it showed a similar banding pattern indicating 100% sensitivity. MPT64 band formation was not detected in any of the 14 isolates indicating 100% specificity. Both PPV & NPV were 100%. All the isolates negative for MPT64 Ag were confirmed as MOTT by conventional bio-chemical PNBA. The H37Rv strain showed a faint band from the 2nd day onwards from inoculation till 3rd day in the earlier Antigen detection experiment. Conclusion: Rapid identification of MTB culture isolate is a pressing need for diagnosis and proceeding to perform drug susceptibility testing. MPT64 TB Ag detection ICT kit is a rapid, reliable method, good substitute for molecular identification methods, and conventional biochemical test which is time-consuming and technically demanding. The early detection of Antigen can be used as an effective tool in diagnosis.

Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.

First Line Anti-Tuberculosis Drugs Resistance Patterns of <i>Mycobacterium tuberculosis</i>Isolates from Newly Diagnosed Cases of Tuberculosis

Introduction: Tuberculosis is a major cause of mortality and morbidity world-wide. Anti-tuberculosis drugs have been used for many decades but resistance to them is now widespread. Globally 5% of tuberculosis cases and in India 3% among new TB cases. This study was planned to know the pattern of first line anti-tuberculosis drug resistance in south Gujarat, Surat region in newly diagnosed patients of tuberculosis. Material and Methods: 350 samples were processed for homogenisation and concentration using 4% NAOH-2.9% trisodium citrate. Processed samples were inoculated in liquid medium that is MGIT (Mycobacterial growth indicator tube). Positive samples for M. tbwere processed further for first line anti-tuberculosis drugs sensitivity testing (DST). Reading was taken by using MicroMGIT system. Result: Out of 350 samples 59 (17%) were positive samples, of which 48 (13%) were M. tb and 11 (3%) were non tuberculous mycobacteria. Out of 48 samples 2% (1 isolate) was resistant to isoniazid and Rifampicin while 2% were monoresistant to isoniazide, 2% monoresistant to streptomycin. No rifampicin monoresistant was detected. Conclusion: Such study may help in control of tuberculosis at regional and national level which would in turn help in planning of measures to control Multi-drug resistance tuberculosis. Continuous surveillance should be applied to know the periodic changing patterns and trend in Drug resistant tuberculosis.

Evaluation of Recombinant <i>Mycobacterium tuberculosis</i>Antigens MPT64, CFP10, and ESAT6 for Delayed-Type Hypersensitivity Responses in Guinea Pigs

The tuberculin Purified Protein Derivative (PPD) skin test is widely used;however, the results are often inaccurate. Positive results can be observed in patients with active tuberculosis (TB) as well as in BCG-vaccinated persons and individuals who are infected with mycobacteria but have not developed the disease. MPT64, an antigen secreted from actively growing Mycobacterium tuberculosis and some strains of M. bovis BCG such as BCG Tokyo and BCG Russia, is immunogenic and elicits delayed-type hypersensitivity (DTH) in guinea pigs and humans. This antigen has been used to develop a new skin test for the diagnosis of active TB infection. Two of the antigens encoded by the M. tuberculosis-specific region of difference 1 (RD1, deleted in M. bovis BCG strains), CFP10 (culture filtrate protein 10) and ESAT6 (early secreted antigenic target-6), also induce M. tuberculosis-specific DTH responses. The aim of this study was to investigate the DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo elicited by three purified recombinant proteins (rMPT64, rCFP10 and rESAT6) compared to those elicited by PPD. In this study genes encoding MPT64, CFP10, and ESAT6 were cloned and expressed as recombinant proteins with the addition of a C-terminal His6 tag for ease of purification by Immobilized Metal ion Affinity Chromatography (IMAC). The recombinant proteins (rMPT64, rCFP10, and rESAT6) were purified to homogeneity and were used to elicit DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo. The results showed that rMPT64 elicits a DTH response comparable to that of PPD in M. bovis BCG Tokyo-vaccinated animals. However, M. tuberculosis-infected animals show less reactivity to rMPT64 than they do to PPD. Although single rCFP10 or rESAT6 did not readily elicit a DTH response in M. tuberculosis-infected animals, combining these antigens with rMPT64 led to an increased DTH response, thus enabling the detection of TB infection.

Estimates of Genetic Variability of <i>Mycobacterium tuberculosis</i>Complex and Its Association with Drug Resistance in Cameroon

The present study investigates the genetic diversity among Mycobacterium tuberculosis complex circulating in the Centre region of Cameroon and analyzes the relationship between genotypes and drug resistance patterns. Spoligotyping was performed by PCR-amplification followed by the reverse hybridization of 298 cultured specimens. Spoligotypes patterns were identified by comparison to reference strains in SPolDB4 database via the MIRU VNTR plus web application. About 97.65% of all tuberculosis (TB) cases were attributed to M. tuberculosis. A total of 65 different profiles were identified. Of these, 40 were represented as Shared Types (ST) while the others were orphans. LAM10_CAM and Haarlem families were the most prevalent genetic families with 51.01% and 14.09% respectively. ST 61, a member of the LAM10_ CAM family formed the largest cluster with 128 (42.95%) isolates. No association was found between genotypes with regard to drug resistance and HIV sero-status. However, there was a significant association between genotypes and age groups. Patients belonging to 15 - 24 and 35 - 44 age groups were more likely infected by LAM10_CAM strains compared to others. The population structure of Mycobacterium tuberculosis complex strains from the Centre region was found to be diverse and the spoligotype 61 of the LAM10_CAM family was highly predominant. Isolates of the LAM10_CAM seem to be not associated with drug resistance.

A new Multilocus Sequence Analysis Scheme for Mycobacterium tuberculosis

Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis （M. tuberculosis） multilocus sequence analysis （MLSA） was established for the phylogenetic and epidemiology analysis. Methods To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods. Results After comparison of the genome, seven high discrimination gene loci （recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR） were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types （STs） were identified. Based on the Hunter-Gaston Index （HGI）, MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types （ST） came into a main cluster, showing the major clonal complexes in those 44 strains. Conclusion MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

Drug Susceptibility Testing of Mycobacteria Isolated from Humans and Cattle from Selected Sites of Ethiopia

Background: The effectiveness of a standard anti-tuberculosis (TB) treatment regimen correlates with in vitro drug susceptibility pattern of the infecting tubercle bacilli. The results of the drug susceptibility tests help select a proper treatment regimen or modify treatment regimen for a better management of patients and surveillance and timely control of the spread of the drug resistant TB in the community. Treatment of drug resistant TB is costly, and the outcomes, including survivorship, can be poor. As the result, the drug susceptibility test has become more important than ever. Objective: To determine the drug-susceptibility pattern of M. tuberculosis and M. bovis isolated from selected sites of Ethiopia. Methods: The conventional indirect L&#246wenstein-Jensen (L-J) proportion method was used to detect the drug susceptibility pattern of 29 isolates of M. tuberculosis and 21 isolates of M. bovis to four anti-TB drugs (streptomycin, rifampicin, isoniazid and ethambutol). Results: Resistance was observed only in M. tuberculosis isolates while all isolates of M. bovis were fully susceptible to the four drugs. Thus, the overall resistance of M. tubeculosis isolates to any of the four drugs was 51.7%. As such, any type of drug resistance was most frequent to streptomycin (41.3%) followed by isoniazid (20.6%) while it was minimal to rifampicin (6.8%) and ethambutol (3.4%). Multidrug resistant TB (MDR-TB) was not detected in the study. Conclusion: This preliminary study showed high level of resistance in M. tuberculosis isolates warranting appropriate use of anti-TB drugs in those sites from where the isolates were obtained.

Evaluation of Increased Sensitivity of Morning Bleach Sample for Detection of Acid Fast Bacilli in Pulmonary Samples

Around 85% of the cases of Tuberculosis (TB) are pulmonary in origin and the routine diagnosis usually depends on sputum microscopy. The conventional direct Ziehl-Nelson (ZN) staining technique has been found to have a low sensitivity. The main objective of the study was to verify whether the bleach concentration method increases the sensitivity of sputum smear microscopy for AFB or not and also to see whether the first single morning sample alone is sufficient and better than the three pooled samples after bleach concentration followed by ZN staining. A total of 365 samples were studied from 131 clinically suspected cases of pulmonary TB which included sputum (112), gastric aspirate (5), endotracheal tube washing (2), and bronchial lavage (12). All these samples were processed for conventional ZN staining and Bleach concentration method followed by ZN staining. An increase in positivity was observed in all the cases after using the bleach concentration method and the most significantly useful was that in the case of first morning sputum samples where it increased from 11.6% to 41.96%. Bleach concentration is a simple, cheap and easily available method and also very safe because it kills the Mycobacteria in the process. Its positivity rate is better as compared to direct conventional ZN staining.

Screening of Several Anti-Infectives for in Vitro Activity against Mycobacterium smegmatis

Aim: To evaluate in vitro the effectiveness of several anti-infective agents alone or in combination against Mycobacterium smegmatis. Method: A convenient stratified sampling method was used to obtain selected anti-infective agents. For individual drug samples, Minimum Inhibitory Concentrations (MIC) were obtained using the agar-well plate diffusion technique. Fractional Inhibitory Concentration Indices (FICI) were calculated for drug combinations using their MIC as obtained from the broth dilution method. Results: Of the thirty (30) anti-infective agents analyzed, ten (10) had MIC equivalent to or better than rifampicin (reference TB drug). Seven (7) drugs had MIC higher than rifampicin, while twelve (12) showed no growth inhibition of M. smegmatis. Analysis of the effect of drug combinations on M. smegmatis indicated that four (4) combinations, including rifampicin/ethambutol showed synergism. One (1) was additive, two (2) were indifferent and one (1) combination showed antagonism. Conclusion: Notable in the results obtained was the high effectiveness of the carbapenems in inhibiting the growth of M. smegmatis. Carbapenems, though not indicated for TB treatment, has a potential of playing a significant role in the treatment of tuberculosis. Also the drug combinations which showed synergism, especially those that involved the macrolide antibiotics, should further be investigated. These results have to be confirmed by in vivo clinical studies to define their roles in tuberculosis treatment.

Taq Man-PCR技术诊断结核病的临床应用研究

目的 探讨TaqMan -聚合酶链反应 (TaqMan -PCR)技术在结核病快速诊断中的临床价值。方法 对 15 5例活动性肺结核患者的痰和外周血、130例结核性胸膜炎患者的胸腔积液和外周血以及 6 1例结核性脑膜炎患者的脑脊液和外周血应用TaqMan -PCR检测结核分支杆菌DNA ,痰、胸腔积液和脑脊液进行抗酸染色涂片检查 ,另外 ,痰标本还进行了BACTEC和改良罗氏培养 ;同期以5 2例肺癌患者的痰和外周血、5 0例恶性胸腔积液患者的胸腔积液以及 33例健康人的外周血作为对照进行TaqMan -PCR检测。 结果 15 5例活动性肺结核患者的痰和外周血、130例结核性胸膜炎患者的胸腔积液和外周血以及 6 1例结核性脑膜炎患者的脑脊液和外周血TaqMan -PCR的阳性率分别为 4 9.0 %和 5 1.6 %、4 5 .4 %和 38.5 %以及 5 0 .8%和 4 2 .6 % ,痰、胸腔积液和脑脊液TaqMan -PCR的阳性率显著高于抗酸染色涂片以及BACTEC和罗氏培养 (P <0 .0 5 )。TaqMan -PCR检测痰、胸腔积液和外周血的特异性分别为 96 .2 %、98%和 96 .5 %。结论 TaqMan -PCR具有较高的敏感性和特异性 ,对结核病的快速诊断具有一定的价值。

噬菌体TM4、Guo1和D29对静止期结核菌裂解作用初步研究

目的研究噬菌体TM4、Guo1、D29能否裂解静止期结核菌。方法采用密闭培养至对数生长期的结核分枝杆菌构建静止期结核菌模型;采用药敏检测、金胺o-尼罗红荧光染色和电镜观察作为检测模型方法;采用most probable number(MPN)法计数作为裂解作用的检测指标。结果密闭培养11个月的静止期结核菌模型构建成功;经不同处理后,MPN计数结果为对照组1100,Guo1组23,TM4组23,D29组600,RFP组673,INH组887;与对照组相比,Guo1组和TM4组菌量明显减少(P<0.01,P<0.01),D29组菌量减少不明显(P=0.05);与RFP组相比,Guo1组和TM4组菌量亦明显减少(P<0.05,P<0.05),D29组菌量减少不明显(P>0.05);Guo1组和TM4组菌量无差异。结论噬菌体TM4和Guo1能够裂解静止期结核菌,噬菌体D29不能裂解静止期结核菌,噬菌体TM4和Guo1裂解能力无差别。

结核杆菌含信号肽的Mtb8.4真核表达质粒的构建及鉴定

目的 对结核杆菌新抗原—带信号肽的Mtb8.4(MS)进行基因克隆 ,构建其真核表达质粒并加以鉴定。 方法 采用聚合酶链反应 (PCR)从结核分枝杆菌H 3 7Rv株基因组中扩增出带信号肽的Mtb8.4(MS)目的基因 ,经HindⅢ和EcoRⅠ消化后 ,与 pcDNA3 .1(+ )载体进行连接重组。 结果 pcDNA3 .1(+ ) -MS真核表达质粒构建完成后 ,用限制性内切酶消化、PCR及DNA测序等多种方法进行鉴定 ,证实其构建成功。 结论 pcDNA3 .1(+ ) -MS真核表达质粒的成功构建 ,为进一步研究该质粒的免疫保护效果 ,了解信号肽序列在MS蛋白表达和分泌过程中所起的作用及制备相应的结核病DNA疫苗奠定了基础。

噬菌体TM4复苏结核休眠菌的初步研究

目的探索噬菌体TM4对结核休眠菌复苏的作用。方法采用密闭培养对数生长期结核菌构建休眠菌模型;采用药敏检测和透射电子显微镜(电镜)观察作为模型检测方法;采用菌落计数和电镜观察作为复苏的检测指标。结果密闭培养180 d休眠菌模型构建成功;培养3 d复苏促进因子(Rpf)E组管底菌量多于空白组和TM4组,培养8 d噬菌体TM4组管底菌量多于空白组,后续观察见TM4组和Rpf E组管底菌量均较前有所增加。混合液培养第1日时,空白组、TM4组和Rpf E组的菌落计数均为0;培养第6日时,菌落计数分别是0、0.7×102和2×104CFU/mL;培养第14日时,菌落计数分别是3.4×10、1.68×107和2.1×109CFU/mL。培养第17日时,电镜观察发现混合物TM4组有大量薄壁结核菌、部分厚壁结核休眠菌和结核菌细胞碎片,Rpf E组满视野薄壁的结核菌,空白组含大量厚壁的结核休眠菌和少数薄壁结核菌。结论噬菌体TM4能够复苏结核休眠菌。

肺结核患者血浆和胸水中抑瘤素-M检测及临床意义

目的探讨血浆和胸水中抑瘤素-M在肺结核中的变化及其临床意义。方法应用双抗体夹心ELISA法检测24例健康体检者、21例活动性肺结核患者血浆和10例结核性胸膜炎胸水中OSM的水平。结果肺结核患者血浆中OSM水平低于健康对照组,差异有统计意义;结核性胸膜炎患者胸水中OSM高于肺结核患者血浆中OSM水平,差异有统计意义。结论OSM是局部病灶炎症反应的重要作用因子,可能参与了肺结核的免疫发病机制。

乙醇对结核分枝杆菌M_r 38000蛋白在大肠杆菌中可溶性表达的促进作用

目的:探讨乙醇促进结核分枝杆菌Mr38000蛋白 的可溶性表达,并获得可溶性的Mr38000重组蛋白.方法: 采用聚合酶链反应(PCR)从结核分枝杆菌H37Rv基因组中 扩增出Mr38000蛋白的编码基因,用限制性内切酶消化后插 入载体pGEX 4T 2中,将重组质粒转化大肠杆菌BL21,在乙 醇添加剂存在下,经IPTG诱导,表达GST 38融合蛋白;聚丙 烯酰胺凝胶电泳(SDS PAGE)分析融合蛋白的相对分子质量 及表达形式,免疫印迹法鉴定融合蛋白的活性.结果:经PCR 扩增后证实为Mr38000蛋白的基因,成功构建了具有正确基 因序列的重组表达载体,在大肠杆菌BL21中诱导表达融合蛋 白约占菌体总蛋白的18%.在乙醇存在下,可溶性目的蛋白的 表达量约是无乙醇时的5倍.结论:在原核表达中乙醇作为一 种添加剂可以促进结核分枝杆菌Mr38000蛋白在大肠杆菌 BL21中的可溶性表达.

结核杆菌含信号肽的Mtb8.4真核表达质粒的构建及在COS-7细胞中的表达

目的:克隆结核杆菌含信号肽的Mtb8·4(MS)基因,导入真核表达载体pcDNA3·1(+),构建成重组质粒pcDNA3·1(+)-MS,并在真核细胞中进行表达。方法:提取人结核杆菌H37Rv株的基因组作为模板,进行PCR圹增,获得含信号肽的Mtb8·4(MS)基因后,与pcDNA3·1(+)载体进行连接重组,用限制性内切酶消化、PCR及DNA序列分析等多种方法进行鉴定;重组质粒转染COS-7细胞48h后,用RT-PCR方法鉴定MS在转录水平的表达情况。结果:pcD-NA3·1(+)-MS真核表达载体构建成功;转染COS-7细胞后,MS在转录水平成功表达。结论:pcDNA3·1(+)-MS真核表达质粒的构建以及MS在COS-7细胞中的成功表达,为进一步研究该真核表达质粒的免疫保护效果及制备结核病pcD-NA3·1(+)-MSDNA疫苗奠定了基础。

结核分枝杆菌新抗原Mtb8.4基因的克隆与真核表达

目的克隆结核杆菌新抗原Mtb8.4基因,导入真核表达载体pcDNA3.1(+),构建成重组质粒pcDNA3.1(+)-Mtb8.4,并在真核细胞中进行表达。方法提取人结核杆菌H37Rv株的基因组作为模板,进行PCR扩增,获得Mtb8.4目的基因后,与pcDNA3.1(+)载体进行连接重组,用限制性内切酶消化、PCR及DNA序列分析等多种方法进行鉴定;重组质粒转染COS-7细胞48h后,用RT-PCR方法鉴定Mtb8.4在转录水平的表达情况。结果pcDNA3.1(+)-Mtb8.4真核表达载体构建成功;转染COS-7细胞后,Mtb8.4在转录水平成功表达。结论pcDNA3.1(+)-Mtb8.4真核表达质粒的构建成功以及Mtb8.4在COS-7细胞中的成功表达,为进一步研究该真核表达质粒的免疫保护效果及制备结核病pcDNA3.1(+)-Mtb8.4DNA疫苗奠定了基础。

基因芯片方法检测耐异烟肼结核分枝杆菌准确性的Meta分析

目的 利用Meta分析方法系统评价基因芯片技术检测耐异烟肼（INH）结核分枝杆菌的准确性。方法 计算机检索The Cochrane Library、SCI、PubMed、EMbase、WanFang Data、CNKI数据库,检索时限均为建库至2015年5月。由2位研究者根据纳入与排除标准筛选文献、提取资料,应用QUADAS条目工具评价纳入研究质量,采用Meta-DiSc 1.4软件对敏感性（SEN）、特异性（SPE）、阳性似然比（＋LR）、阴性似然比（-LR）、诊断比值比（DOR）进行异质性检验和合并分析并绘制受试者工作特征（SROC）曲线、计算曲线下面积（AUC）。结果 共纳入15篇符合要求文献,分析3 967株经传统药敏试验鉴定结核分枝杆菌菌株,其中耐INH菌株1 147株、敏感菌株2 820株。基因芯片技术检测耐INH结核分枝杆菌的SEN_（并发）为0.82[95%CI（0.79~0.84）]、SPE_（并发）为0.97[95%CI（0.97~0.98）]、＋LR为21.81[95%CI（12.12~39.25）]、-LR为0.19[95%CI（0.15~0.24）]、DOR_（并发）为136.55[95%CI（70.66~263.89）],AUC为0.94。结论 通过循证学方法系统评价基因芯片技术检测耐INH结核分枝杆菌结果表明,其具有较好的诊断价值;与传统药敏试验相比基因芯片技术操作简单、检测快速,对耐药基因检出率较高,但费用较贵,成本较高。

结核分枝杆菌稳定L型耐乙胺丁醇embB基因的研究

目的探讨结核分枝杆菌L型形成乙胺丁醇耐药性的分子机制。方法采用非高渗分离培养法在不含乙胺丁醇与含乙胺丁醇的液体培养基内诱导形成结核分枝杆菌L型,过滤传代后获得稳定L型纯培养物。通过nPCR扩增、PCR-SSCP及PCR-DS技术分别对自发形成与诱导形成的结核分枝杆菌稳定L型耐乙胺丁醇embB基因进行检测和分析。结果自发形成及药物诱导形成的结核分枝杆菌稳定L型embB基因的nPCR及PCR-SSCP结果显示,与其亲代细菌型一致的DNA条带和带型;测序结果显示稳定L型embB基因序列与其亲代细菌相同没有发生改变,其结果与PCR-SSCP分析结果一致。结论 PCR-SSCP和PCR-DS技术可用于结核分枝杆菌L型耐乙胺丁醇基因的检测;无论是自发形成或药物诱导形成的稳定L型embB基因未发生突变,提示结核分枝杆菌L型对乙胺丁醇的耐药性同embB基因突变无关,细胞壁缺陷变异可能是导致结核分枝杆菌产生乙胺丁醇耐药性的一个重要机制。

结核分枝杆菌含信号肽的Mtb8.4基因的克隆及真核表达质粒的构建(英文)

目的对结核杆菌新抗原—带信号肽的Mtb8.4(MS)进行基因克隆,构建其真核表达质粒并加以鉴定。方法采用聚合酶链反应(PCR)从结核分枝杆菌H37Rv株基因组中扩增出带信号肽的Mtb8.4(MS)目的基因,经HindⅢ和EcoRⅠ消化后,与pcDNA3.1(+)载体进行连接重组。结果pcDNA3.1(+)-MS真核表达质粒构建完成后,用限制性内切酶消化、PCR及DNA测序等多种方法进行鉴定,证实其构建成功。结论pcDNA3.1(+)-MS真核表达质粒的成功构建,为进一步研究该质粒的免疫保护效果,了解信号肽序列在MS蛋白表达和分泌过程中所起的作用及制备相应的结核病DNA疫苗奠定了基础。