长臂同源多聚酶链反应法构建变形链球菌comD基因同源重组DNA片段

目的构建变形链球菌UAl59密度感应相关的comD基因同源重组DNA片段,为利用同源重组原理构建基因功能丧失菌株做准备。方法通过NCBI基因数据库获取变形链球菌的DNA序列,利用聚合酶链反应技术分别扩增变形链球菌UA159comD基因上、下游片段及抗红霉素基因片段,再通过长臂同源多聚酶链反应将这3个片段连接起来,形成同源重组DNA片段。结果经过PCR反应和琼脂电泳分析,得到了一个碱基数为3个单片段总和的连接片段,测序结果显示连接片段为预期的comD同源重组片段。结论成功构建了变形链球菌UA159comD基因同源重组DNA片段,可直接用于细菌转化构建comD基因缺陷菌株。     

替代失活法构建变形链球菌LuxS基因缺陷株

目的 LuxS基因是变形链球菌生物膜早期形成过程中的关键基因,构建该基因的缺陷菌。方法采用长臂同源多聚酶链反应（LFH-PCR）方法构建含红霉素耐药基因片段的LuxS基因上、下游同源序列的连接片段,转化到变形链球菌中,在红霉素的平板上筛选缺陷菌株,并采用PCR鉴定。结果对变形链球菌LuxS基因缺陷菌株进行PCR和DNA序列测定分析证实构建成功。结论成功构建出变形链球菌LuxS基因的缺陷菌株,为后期针对变形链球菌LuxS基因的相关研究奠定基础。     

变形链球菌耐氟菌株耐酸相关基因ropA的克隆及蛋白表达

克隆并表达变形链球菌耐氟菌株耐酸相关基因ropA。以变形链球菌UA159的耐氟菌株全基因组为模版,PCR扩增ropA基因并与p MD-18T克隆载体连接构建重组克隆质粒,测序鉴定。Bam HⅠ、HindⅢ双酶切重组克隆质粒,回收目的基因片段并与p Pro EX HTa表达载体通过粘性末端连接构建重组表达质粒,转化入大肠埃希菌感受态细胞DH5α,IPTG诱导,SDS-PAGE检测Rop A表达量。测序结果为变形链球菌UA159的耐氟菌株ropA基因碱基序列与亲代菌株UA159完全一致。SDS-PAGE结果显示IPTG成功诱导Rop A蛋白表达,并且随诱导时间的延长蛋白表达增多。变形链球菌UA159耐氟菌株的耐酸相关基因ropA未发生突变,说明变形链球菌耐氟菌株耐酸性增强不是由ropA碱基序列的改变导致。本研究成功诱导Rop A蛋白表达,为后续研究Rop A蛋白功能奠定了基础。     

鼠伤寒沙门菌spvBC基因编辑株的构建

利用λRed重组系统和pBAD原核表达载体构建鼠伤寒沙门菌spvBC质粒毒力基因修饰菌株,为深入探究沙门菌毒力基因spv的功能和致病机制及宿主抗感染免疫提供工具菌。以pKD4为模板,PCR扩增含spvBC同源臂的卡那霉素抗性基因以构建同源打靶片段,再将其电转入含有质粒pKD46的鼠伤寒沙门菌中进行同源重组,随后将质粒pCP20电转导入阳性转化子,消除卡那霉素抗性基因,PCR鉴定敲除株的构建。PCR扩增含酶切位点的spvBC基因片段,扩增产物与原核表达载体pBAD/gⅢ分别双酶切后连接构建pBAD-spvBC重组质粒,PCR筛选阳性菌落并测序鉴定。将构建成功的pBAD-spvBC重组质粒电转导入spvBC敲除株中,Western blot测定不同浓度L-阿拉伯糖诱导SpvB和SpvC蛋白表达情况。PCR结果表明鼠伤寒沙门菌spvBC基因敲除成功;PCR及测序结果表明pBAD-spvBC重组质粒构建成功,Western blot结果表明13 mmol/L L-阿拉伯糖可诱导SpvB和SpvC蛋白正常表达。λRed重组系统可用于沙门菌质粒上大片段基因的敲除,pBAD原核表达载体可用于沙门菌质粒上大片段基因的回补,丰富了细菌质粒的基因修饰和编辑策略。     

大肠杆菌胸腺嘧啶合成酶thyA缺失突变菌株的构建

大肠杆菌K12 DY330菌株的染色体上整合有一种新型的同源重组系统——缺陷型λ原噬菌体同源重组系统。以DY330为出发菌,通过同源重组构建大肠杆菌thyA-株DY330TI,其基因组特点是：thyA基因除保留N端的1～49氨基酸残基相对应的必需核苷酸序列外,将其余部分全部缺失;此外,还敲除了DY330TI中与缺陷型λ原噬菌体同源重组功能相关的基因,从而尽可能避免了通过同源重组产生回复突变的可能性。通过大肠杆菌thyA基因对该突变株的转化实验,检测转化子的回复突变率,进一步证实该突变株的突变性状稳定,为构建以thyA为选择标志的大肠杆菌染色体质粒平衡致死系统提供了合适的缺陷型宿主菌。     

per基因突变对布鲁菌稳定性的影响

目的:构建过骨胺酸合成酶基因(per)敲除的布鲁菌,研究per基因对布鲁菌株稳定性的影响。方法:构建p MD19-T:kan敲除载体,电击转化至流产布鲁菌,体内同源重组敲除per基因,分析per基因敲除对布鲁菌遗传特征和稳定性的影响。结果:构建了per基因敲除的流产布鲁菌株,per缺失突变布鲁菌连续传代培养后,测序分析未发现回复突变,品红及硫堇培养鉴定符合布鲁菌特征。结论:布鲁菌基因组per基因的缺失突变不影响布鲁菌的遗传稳定性,为研制布鲁菌per基因减毒突变疫苗提供了依据。     

新生隐球菌转录共激活因子MBF1基因的鉴定与敲除

目的构建新生隐球菌转录共激活因子MBF1基因缺陷菌株。方法采用套叠PCR方法 ,构建含有抗性基因NEO以及靶基因上下游同源DNA片段的基因敲除框,通过基因枪将重组片段转化入新生隐球菌,应用PCR筛选和DNA序列测序方法对基因突变株进行鉴定与验证。结果成功构建了新生隐球菌基因突变株mbf1裣。结论通过基因突变株mbf1裣的构建,为深入研究新生隐球菌转录辅助因子Mbf1的功能机制奠定基础。     

利用λRed重组系统敲除鼠伤寒沙门氏菌sopB基因

利用λRed重组系统敲除鼠伤寒沙门氏菌LT2的(Salmonella enterica serovar typhimurium LT2,S.typhimurium LT2)sopB基因。以pKD4质粒为模板,扩增得到中间带有卡那霉素抗性基因且两端各带有59 bp分别与sopB基因上下游序列同源的同源打靶片段,将其转化至表达Red重组酶的S.typhimurium LT2感受态细胞中;在抗生素压力和λRed重组系统帮助下,同源片段和菌体sopB基因发生同源重组,通过卡那霉素筛选得到带有抗性标记的阳性重组菌;转入重组酶表达质粒pCP20以除去抗性标记,得到保留单一FRT位点的突变菌株;利用PCR技术鉴定重组菌,并通过检测沙门氏菌效应蛋白SopB的分泌以及沙门氏菌感染HeLa细胞后pAKT的激活反应来鉴定sopB基因是否被敲除。构建的ΔsopB突变菌株失去了分泌SopB蛋白的能力,且不能够像野生型菌株那样在感染HeLa细胞的过程中激活pAkt。本研究获得了S.typhimurium LT2的sopB基因缺失突变株,为沙门氏菌感染宿主过程中SopB的功能研究提供工具,同时也为进一步探索其他类型细菌的基因敲除提供了线索。     

λRed重组系统用于沙门菌质粒毒力基因spvC敲除株的构建

[目的]利用λRed重组系统敲除沙门菌质粒毒力基因spvC。[方法]首先以质粒p KD4为模板,扩增得到两侧含spvC同源臂、中间为卡那霉素抗性基因的线性DNA片段。再将此线性片段电转入具重组功能的感受态沙门菌菌株,发生重组后,卡那霉素平板筛选阳性转化子。最后利用表达FLP重组酶的质粒p CP20,将FRT位点之间的卡那霉素抗性基因消除,用PCR鉴定。Western Blot检测野生沙门菌和spvC敲除株感染的He La细胞ERK磷酸化水平。[结果]沙门菌质粒毒力基因spvC敲除株构建成功,spvC敲除株感染的He La细胞内ERK磷酸化水平升高。[结论]成功构建沙门菌质粒毒力基因spvC敲除株,验证了spvC基因的功能。     

假想的脂蛋白连接酶对肺炎链球菌毒力的影响

【目的】探索假想脂蛋白连接酶(putative lipoate-protein ligase,LPL)对肺炎链球菌毒力的影响。【方法】采用长臂同源多聚酶链式反应(LFH-PCR)的方法失活lpl基因,通过PCR、测序鉴定缺陷菌株,采用细胞实验比较缺陷菌和野生菌对宿主细胞的粘附能力,并通过动物实验观察lpl基因缺陷后菌株毒力的变化。【结果】小鼠毒力实验表明野生菌株和缺陷株半数致死时间均为12h,两者比较无统计学差异;缺陷菌在对宿主细胞的粘附能力明显高于野生菌株(P0.01);体外荚膜染色实验表明,野生菌和缺陷菌均有荚膜。【结论】实验结果提示lpl基因对细菌粘附宿主细胞有抑制作用,但不影响其腹腔感染小鼠的能力。     

框内缺失突变法构建变形链球菌comE基因突变株

变形链球菌的种内密度感应系统由com基因家族编码控制。膜受体ComD接受密度感应信号后激活菌体内的反应调节子ComE,ComE作为启动子可以调节一系列相关基因的表达。应用框内缺失突变法（in-frame deletion）,通过两次同源性重组,成功构建了变形链球菌comE基因突变株IFD140ΔcomE。由于框内缺失突变没有引入任何的遗传标记物,所以有效地避免了传统的基因失活方法——插入重复(insertion duplication) 和等位交换(allelic exchange),所导致的下游基因极性效应（polar effect）。经过PCR、测序分析及RT-PCR,证实IFD140ΔcomE仅在comE基因内部缺失了717bp的片段,未引入任何外源性DNA,且comE下游的comD基因可以正常转录,无极性效应产生。对IFD140Δcom表型特征的研究发现,在液体培养基中IFD140Δcom更易发生沉淀性生长和贴壁性生长。菌体呈长链状排列。IFD140ΔcomE的成功构建,为进一步研究变形链球菌种内密度感应系统奠定基础。     

变形链球菌反应调控蛋白ComE结合序列的初步筛选

目的：从变形链球菌基因组中筛选反应调控蛋白ComE结合的核酸序列。方法：⑴提取变形链球菌UA159 基因组,进行超声剪切处理。以基因组片段为模板,用随机引物进行延伸,切胶纯化和PCR 扩增后获得基因组文库。⑵应用基因组 SELEX 技术,以ComE 蛋白为靶物质,与变形链球菌基因组文库共同孵育,循环筛选8轮。筛选产物TA克隆后送去测序,测序结果进行生物信息学分析。⑶将分析得到的2个序列分别克隆入pFW5-luc载体中,然后分别重组到变形链球菌UA159野生株和comE突变株的基因组中,采用RT-PCR的方法检测luc片段的表达。结果：⑴获得了变形链球菌UA159 的基因组文库,片段范围约为100～300bp。⑵随机挑取54个克隆送去测序,经生物信息学分析和RT-PCR初步验证,最终获得1个ComE 蛋白可能的结合序列。结论：采用基因组 SELEX 技术,筛选反应调控蛋白ComE可能的结合序列,并进行了初步验证,为后续进行变形链球菌反应调控蛋白ComE调控机制的研究奠定了基础。     

A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.     

BrpA is involved in regulation of cell envelope stress responses in Streptococcus mutans

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by Streptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of brpA is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a Galleria mellonella (wax worm) model, BrpA deficiency was shown to diminish the virulence of S. mutans OMZ175, which, unlike S. mutans UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of S. mutans.     

The Streptococcus mutans vicX gene product modulates gtfB/C expression, biofilm formation, genetic competence, and oxidative stress tolerance

Streptococcus mutans is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of S. mutans. In this study, we focused on the vicX gene of the vicRKX tricistronic operon. To characterize vicX, we constructed a nonpolar deletion mutation in the vicX coding region in S. mutans UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of vicX (P < 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in S. mutans. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly, vicX-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (gtfB/C) expression compared with the expression in the wild type (P < 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an S. mutans GS5-derived vicX null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking gtfB and/or gtfC. Also, disruption of vicX reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (P < 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the vicX gene product that can impact S. mutans pathogenicity.     

新生隐球菌COP9复合体基因CSN6的敲除与鉴定

目的构建新生隐球菌COP9复合体蛋白元件Csn6的基因同源重组敲除框,并通过基因枪转化系统敲除CSN6基因。方法应用生物信息学方法获得COP9复合体蛋白元件的基因信息,采用套叠PCR的方法,构建包含报告基因NEO和CSN6基因ORF两侧上下游同源DNA片段的同源重组框。应用基因枪将其转化入新生隐球菌感受态细胞,通过PCR和DNA测序对遗传霉素(G418)耐受的阳性克隆子进行筛选与验证。结果成功构建了新生隐球菌基因突变株csn6裣。结论 COP9复合体亚基CSN6基因突变株的构建,为今后新生隐球菌COP9复合体的分子致病机制研究奠定基础。