胞壁表达HSP65-IL-2融合蛋白的重组耻垢分枝杆菌的构建和鉴定

目的：获得胞壁表达结核分枝杆菌热激蛋白65（HSP65）和人白细胞介素2（IL-2）融合蛋白的重组耻垢分枝杆菌。方法：将HSP65和IL-2融合基因克隆入大肠杆菌-卡介苗（E．coli-BCG）穿梭质粒pCW,构建成重组质粒HSP65-IL-2-pCW,电穿入耻垢分枝杆菌,经潮霉素抗性筛选和PCR方法选取阳性克隆;用间接免疫荧光法对重组耻垢分枝杆菌进行表型鉴定;用重组耻垢分枝杆菌免疫BALB／c小鼠,检测其诱导的抗体水平。结果：筛选获得的重组耻垢分枝杆菌增殖特性与普通耻垢分枝杆菌无明显区别;与抗人的IL-2抗体和HSP65抗体均可形成免疫印迹条带;间接荧光染色后,可见细菌表面有极强的绿色荧光;重组耻垢分枝杆菌免疫BALB／c小鼠2周后,小鼠血清中HSP65抗体平均滴度为1：4000,而生理盐水组的抗体几乎为阴性。结论：结核分枝杆菌HSP65和人IL-2融合基因在耻垢分枝杆菌胞壁获得表达,有望为结核病的预防提供有效的疫苗。     

结核杆菌活动期高表达基因Rv1776c的克隆及其在耻垢分枝杆菌中的表达

目的构建表达结核分枝杆菌Rv1776c基因的重组耻垢分支杆菌,并鉴定该基因在重组耻垢分支杆菌中的活性。方法采用PCR技术克隆结核分枝杆菌Rv1776c基因,构建大肠埃希菌-分支杆菌穿梭表达质粒pMV-Rv1776c,通过酶切和测序鉴定其正确性,用电穿孔法将重组质粒转染到耻垢分支杆菌mc^2155中。以SDS-PAGE及Western blot检测证实Rv1776c蛋白在重组耻垢分支杆菌内的表达。结果重组耻垢分支杆菌构建成功,生长曲线说明重组质粒不会影响耻垢分支杆菌的体外生长;SDSPAGE及Western blot检测证实Rv1776c在耻垢分枝杆菌内表达出相对分子量约56kD的Rv1776c蛋白。结论成功构建了Rv1776c基因的穿梭质粒pMV-Rv1776c,且该质粒在耻垢分枝杆菌内具有生物活性,为进一步研究其表达产物的功能提供基础。     

结核杆菌eis基因的克隆及其在耻垢分枝杆菌中的表达

目的:构建结核分枝杆菌eis基因的穿梭表达载体,鉴定其在重组耻垢分枝杆菌中的生物活性。方法:采用PCR技术克隆结核分枝杆菌eis基因,构建大肠杆菌-分枝杆菌穿梭表达载体pMV-eis,经酶切和测序鉴定其正确性,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc²155中,采用SDS-PAGE和Western blot检测eis基因在耻垢分枝杆菌中的表达。结果:成功构建结核杆菌eis基因穿梭表达载体pMV-eis;生长曲线说明重组质粒不会影响耻垢分枝杆菌的体外生长;SDS-PAGE 和Western blot检测证实eis在耻垢分枝杆菌中可表达出相对分子量约42kDa的Eis蛋白。结论:成功构建了eis基因穿梭表达质粒pMV-eis,且该重组质粒在耻垢分枝杆菌中具有生物活性,为下一步研究表达产物Eis的功能奠定了一定基础。     

大肠杆菌-分枝杆菌分泌型穿梭表达质粒pBCG-SP-HSP65的构建及人结核杆菌HSP65的表达

用PCR技术从Bacillus Calmetteguérin(BCG)基因组中扩增出抗原85B(Ag85B)的信号肽(SP)DNA序列,从pCMVMTHSP65质粒中扩增出人结核杆菌HSP65全长基因。利用DNA重组技术将以上两个片段插入质粒pBCG2100的人结核杆菌HSP70启动子下游,构建成分泌型原核穿梭表达质粒(pBCGSPHSP65)。酶切鉴定、PCR和测序分析结果均表明分泌型原核穿梭表达质粒pBCGSPHSP65构建成功。利用电穿孔将该质粒转入耻垢分枝杆菌(Mycobacterial smegmatis, MS)中,用卡那霉素筛选出阳性重组子。经热诱导后用SDS-PAGE观察到在耻垢分枝杆菌中65kD蛋白占总蛋白的20%,而在重组耻垢分枝杆菌表达的65kD蛋白占菌体总蛋白的34.46%,占裂解物上清总蛋白的68.56%,表明重组的HSP65基因能在耻垢分枝杆菌中高效表达,表达的蛋白大部分以可溶状态存在。通过Westernblot证实分泌的该蛋白能与结核杆菌HSP65的抗体特异性结合,说明该重组蛋白具有HSP65的生物学活性。     

结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达

目的：在耻垢分枝杆菌中表达重组结核杆菌DnaA蛋白并对表达产物进行鉴定。方法：用PCR的方法扩增结核杆菌dnaA基因并克隆至表达载体pMF406中,构建重组大肠杆菌-分枝杆菌穿梭质粒pMF-dnaA。经双酶切及测序鉴定后,用电转化的方法将重组质粒转至耻垢分枝杆菌mc2155中。用0.02%乙酰胺诱导重组耻垢分枝杆菌,对表达产物进行SDS-PAGE和Western blotting检测和鉴定。结果：重组耻垢分枝杆菌构建成功,SDS-PAGE及Western blotting结果显示该重组耻垢杆菌可以实现结核杆菌DnaA蛋白的同源高效表达。结论：结核杆菌DnaA蛋白的同源表达为结核杆菌DNA复制机制的研究奠定了基础。     

ESAT6-CFP10融合蛋白基因的克隆及其在耻垢分枝杆菌中的表达

目的:构建能表达ESAT6-CFP10融合蛋白的重组耻垢分枝杆菌疫苗。方法:以结核杆菌H37Rv基因组DNA为模板,克隆cfp10基因,将其插入到pGEM-T-easy载体后与esat6基因连接,构建含有esat6和cfp10融合基因的重组载体pGEM-e6c10。酶切消化pGEM-e6c10重组质粒,将esat6-cfp10基因亚克隆到pDE22分泌表达穿梭载体,以电穿孔方法将重组质粒转化至耻垢分枝杆菌。PCR筛选到的阳性克隆经温度诱导后,SDS-PAGE和Western印迹检测表达蛋白的正确性。结果:可表达出相对分子质量约23000的ESAT6-CFP10融合蛋白。Western印迹证明重组蛋白有较好的免疫原性。结论:获得能表达ESAT6-CFP10融合蛋白的重组耻垢分枝杆菌,为结核病的预防奠定了一定的基础。     

结核分枝杆菌eis基因对鼠巨噬细胞Raw264．7白噬影响的研究

目的探讨结核分枝杆菌eis基因对巨噬细胞自噬的影响。方法将鼠巨噬细胞Raw264．7以自噬体荧光表达质粒GFP-LC3转染,将含eis基因的重组耻垢分枝杆菌MS—pmv261-eis与不含eis基因的耻垢分枝杆菌MS—pmv261分别感染宿主巨噬细胞,透射电镜下观察自噬小体形成情况,荧光显微镜下观察自噬荧光并计数,Westernblot检测如基因表达的蛋白及自噬蛋白LC3-Ⅱ的表达水平。结果结核分枝杆菌eis基因可抑制感染宿主细胞自噬小体的形成,并显著抑制自噬荧光小点形成（P〈0．05）,显著降低了自噬蛋白LC3-Ⅱ表达水平。结论结核分枝杆菌e曲基因对Raw264．7细胞自噬有抑制作用。     

基于光激活定位显微镜的膜蛋白鉴定技术

病原菌膜蛋白的原位标记和定位追踪是一项非常繁琐的工作。为了建立一种可以用于快速鉴定膜蛋白、且分辨率达到纳米级别的膜蛋白荧光定位技术,将结核分枝杆菌外膜蛋白OmpA与具有光敏活性的蛋白mEos2m在无致病性的耻垢分枝杆菌中进行融合表达。重组菌固定在载玻片上后,利用405 nm激光激活mEos2m。利用普通荧光体视显微镜、正置荧光显微镜和超分辨率光激活定位显微镜观察OmpA-mEos2m,分析融合蛋白在细菌内的分布情况,并捕获光激活蛋白在细胞膜上释放的光子信号。通过超分辨率光激活定位显微镜,发现OmpA-mEos2m融合蛋白在细胞膜上呈"带"状环绕分布,这是观察到膜蛋白在细胞膜上定位的最直接证据。mEos2m与OmpA融合表达后,并不改变OmpA的膜蛋白属性和定位特征。因此可以将mEos2m用于其他非多聚体膜蛋白的融合和定位研究。可以应用非致病性的耻垢分枝杆菌作为模型,采用高分辨率活菌成像技术,研究致病性的结核分枝杆菌蛋白结构、定位和功能。这是目前为止国内采用光激活定位显微成像技术研究膜蛋白的首次报道。     

耻垢分枝杆菌MSMEG_6281 过表达对菌株生长和形态的影响

【目的】耻垢分枝杆菌(Mycobacterium smegmatis mc2155,mc2155)MSMEG_6281为结核分枝杆菌自溶素Rv3717的同源蛋白,通过建立过表达MSMEG_6281的耻垢分枝杆菌菌株,推测该蛋白对耻垢分枝杆菌肽聚糖代谢的影响。【方法】利用RT-PCR方法检测乙胺丁醇(Ethambutol,EMB)作用后MSMEG_6281基因的表达变化;以耻垢分枝杆菌基因组DNA为模板,采用PCR技术克隆MSMEG_6281基因,构建分枝杆菌表达质粒p VV16-MSMEG_6281,进一步建立MSMEG_6281过表达的耻垢分枝杆菌菌株;利用生长曲线检测MSMEG_6281过表达对耻垢分枝杆菌生长的影响;利用扫描电子显微镜分析MSMEG_6281过表达引起的耻垢分枝杆菌形态变化。【结果】EMB处理引起MSMEG_6281基因表达上调;构建了过表达MSMGE_6281的耻垢分枝杆菌菌株(mc2155/p VV16-MSMEG_6281);过表达MSMGE_6281的耻垢分枝杆菌生长缓慢,菌体形态由短杆状转变为长杆状。【结论】MSMGE_6281的过表达可改变耻垢分枝杆菌形态。MSMGE_6281的功能与细胞壁肽聚糖水解相关,在mc2155细胞壁形态维持方面发挥重要作用。     

复苏促进因子结合蛋白A的原核表达及对耻垢分枝杆菌的促生长作用

目的:在大肠埃希氏菌中表达结核分枝杆菌复苏促进因子结合蛋白A(resuscitation-promoting factor-interacting protein A,RipA),观察该蛋白对耻垢分枝杆菌的促生长作用.方法:采用PCR方法,从结核分枝杆菌H37Rv的DNA中扩增出编码RipA蛋白的Rv1477基因,测序正确后克隆入原核表达载体pProEx HTa,构建重组表达质粒pProEx HTa-RipA.以重组质粒转化大肠杆菌DH5a,筛选阳性重组菌株,经异丙基硫代-β-D半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside IPTG)诱导RipA表达,在变性条件下对目的蛋白进行亲和层析纯化.将纯化的RipA蛋白加入到耻垢分枝杆菌中,分光光度法测定细菌的A600,观察RipA蛋白的促生长作用.结果:成功扩增了Rv1477基因,并克隆于表达载体pProEx HTa中,经酶切鉴定获得阳性克隆.经诱导在大肠杆菌中表达出相对分子量为52kDa的目的蛋白,Western-blot结果显示该蛋白与6x His单抗有特异性的反应条带.采用Ni+-NTA柱可获得纯化的目的蛋白.10%M的RipA蛋白可显著促进耻垢分枝杆菌休眠菌的复苏和生长.结论:Papa蛋白在大肠杆菌中成功表达,并能有效促进耻垢分枝杆菌的生长.     

鸡传染性喉气管炎病毒gB基因的克隆及其在耻垢分枝杆菌中的表达

以ILTV基因组为模板 ,利用PCR特异扩增出gB基因 ,定向克隆到中间质粒载体pY_α ,构建了中间质粒pY_α_gB。然后以中间质粒pY_α_gB为模板 ,扩增出含有人结核分枝杆菌启动子hsp70基因和堪萨斯分枝杆菌α信号肽基因的hsp_α_gB片段 ,回收补平后与穿梭表达载体pRR3平端连接 ,从而构建大肠杆菌_分枝杆菌穿梭表达质粒pR_α_gB。再将其电转化至耻垢分枝杆菌M .smegmatismc2 15 5 ,ELISA检测表明重组菌株M .smegmatismc2 15 5 (pR_α_gB)的表达产物具有很好的反应原性。Westernblot检测说明gB基因在分枝杆菌中获得了表达并具有良好的免疫原性。鸡胚中和试验结果表明该重组菌株可以中和 1个剂量EID50 的ILTV强毒 ,能够保护SPF鸡胚抵抗强毒攻击     

Generation of useful insertionally blocked sterol degradation pathway mutants of fast-growing mycobacteria and cloning, characterization, and expression of the terminal oxygenase of the 3-ketosteroid 9alpha-hydroxylase in Mycobacterium smegmatis mc(2)155

Integration of the pCG79 temperature-sensitive plasmid carrying Tn611 was used to generate libraries of mutants with blocked sterol-transforming ability of the sterol-utilizing strains Mycobacterium smegmatis mc(2)155 and Mycobacterium phlei M51-Ept. Of the 10,000 insertional mutants screened from each library, 4 strains with altered activity of the sterol-degrading enzymes were identified. A blocked 4-androstene-3,17-dione-producing M. phlei mutant transformed sitosterol to 23,24-dinorcholane derivatives that are useful starting materials for corticosteroid syntheses. A recombinant plasmid, pFJ92, was constructed from the genomic DNA of one of the insertional mutants of M. smegmatis, 10A12, which was blocked in 3-ketosteroid 9alpha-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9alpha-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9alpha-hydroxylase in M. smegmatis mc(2)155 and has domains typically conserved in class IA terminal oxygenases. Escherichia coli containing the gene could hydroxylate the steroid ring at the 9alpha position.     

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.     

携带PTEN基因的重组腺病毒表达载体构建的研究

构建携带抑癌基因PTEN(Phosphatase and temin homolog deleted on chromosome ten)的重组腺病毒表达裁体,为研究PTEN的功能和作用机制奠定基础.采用RT-PCR法从大鼠海马神经元扩增目的基因PTEN,克隆人含绿色荧光蛋白(Green fluorescence protein),GFP基因的pAdTrack-CMV穿梭质粒,在含有腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌内进行同源重组;获得重组腺病毒质粒,经Pacl线性化后,转染AD293细胞.结果表明,感染腺病毒载体的AD293细胞表达GFP基因,随着时间逐渐增强,并且出现明显的细胞病变效应(Cytopathic effect,CPE),经PCR对传代的Ad-PTEN分析证实得到目的基因.成功构建了携带PTEN基因的腺病毒表达载体,为研究PTEN的功能和作用机制奠定了基础.     

SOCS3基因重组腺病毒的构建及其在猪脂肪细胞中的表达

本研究旨在构建细胞因子信号转导抑制因子3(Suppressor of cytokine signaling 3,SOCS3)的重组腺病毒表达载体,获得有感染性的病毒颗粒。以pcDNA3-SOCS3质粒为模板扩增SOCS3基因,将其亚克隆至腺病毒穿梭载体pAdTrack-CMV,经测序验证后,重组的穿梭质粒用PmeI酶切线性化后转化到BJ5183感受态细菌中与其内的骨架载体pAdEasy-1进行同源重组,获得的重组质粒pAd-SOCS3,经PacI线性化后转染至HEK293细胞中进行包装和扩增,纯化后用TCID50法测定病毒滴度。以重组的病毒感染原代培养的猪脂肪细胞后,荧光显微镜下观察报告基因GFP的表达,RT-PCR和Western blotting检测细胞内SOCS3 mRNA和蛋白的表达。重组腺病毒载体pAd-SOCS3经酶切及PCR鉴定正确,病毒滴度为1.2×109PFU/mL;感染原代培养的猪脂肪细胞后,荧光显微镜观察可见报告基因GFP的表达;RT-PCR和Western blotting检测到细胞中SOCS3 mRNA和蛋白的表达显著提高。本研究成功构建了SOCS3基因的重组腺病毒,感染原代培养的猪脂肪细胞可稳定表达SOCS3蛋白,为深入研究SOCS3的功能奠定了基础。     

Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis

A method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.     

Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis.

The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis. Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP). A 160-fold variation of GFP expression levels in M. smegmatis was achieved by combining three promoters with different copy numbers of the vectors. An efficient energy transfer between BFP and GFP in M. smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors. Thus, these vectors will be valuable for all strategies where co-expression of proteins in M. smegmatis is needed, e.g. for constructing a two-hybrid system or for deleting essential genes.