鼠伤寒沙门菌rtsB基因缺失株的构建及其生物学特性

【背景】鼠伤寒沙门菌(Salmonella typhimurium)是一种重要的人畜共患病原菌,严重危害养殖业及人类健康。调控蛋白在病原菌的生存及感染过程中发挥重要作用。【目的】构建鼠伤寒沙门菌调控基因rtsB缺失株和互补株,分析调控蛋白RstB对鼠伤寒沙门菌生物学特性和致病性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌SAT52的rtsB基因缺失株,并利用互补质粒构建互补株。然后比较分析野生株SAT52、缺失株?rtsB和互补株C?rtsB的生长特性、运动性、生物被膜形成能力、黏附入侵能力、胞内存活能力及致病性的差异。【结果】缺失rtsB基因不影响SAT52的生长速度,但导致运动能力增强,生物被膜形成能力减弱。细胞感染试验结果表明,rtsB基因有助于鼠伤寒沙门菌对Hela细胞的黏附入侵及RAW264.7细胞内的存活。动物试验结果表明rtsB基因缺失显著降低鼠伤寒沙门菌的致病力。【结论】rtsB基因在鼠伤寒沙门菌感染过程中发挥重要作用,可为阐释鼠伤寒沙门菌的致病机制提供参考。     

鸡白痢沙门菌ipaJ基因的克隆与鉴定

摘要：【目的】从鸡白痢沙门菌C79-13中克隆ipaJ基因,体外表达该蛋白后进行免疫原性分析。【方法】鸡白痢沙门菌C79-13与肠炎沙门菌50041进行抑制差减杂交后获得的片段PEA2、PE31和PE44与猪霍乱沙门菌C500疫苗株pSFD10质粒上ipaJ基因高度同源,拼接后获得了鸡白痢沙门菌完整的ipaJ基因序列。从鸡白痢沙门菌中克隆出ipaJ基因并将其构建到原核表达载体pET-30a(+)上,Western-blot检测体外表达蛋白的免疫原性,同时检测了该基因在鸡白痢沙门菌分离株中的分布。【结果】从鸡白痢沙门菌中克隆了大小为840 bp的ipaJ基因序列,并获得了体外原核表达的大小为37 kDa融合蛋白。该蛋白可与鸡白痢沙门菌阳性血清反应。PCR结果显示该基因广泛存在于鸡白痢沙门菌菌株中。 【结论】本文首次报道和克隆了鸡白痢沙门菌ipaJ基因,并证明了IpaJ蛋白具有免疫原性。     

肠道沙门氏菌O-抗原多糖的研究进展

O-抗原是由多糖重复单元组成的多聚糖,表达于细菌的外膜,具有多样性,是划分沙门菌血清型的重要依据。O-抗原多糖由多基因协同作用而合成,这些基因在沙门菌基因组上成簇存在,形成O-抗原基因簇。O-抗原多糖也是重要的毒力因子,在沙门菌入侵宿主、体内存活、定殖等致病过程中均发挥着重要的作用。此外,O-抗原还是沙门菌主要的保护性抗原,能激发宿主产生高水平抗体并发挥免疫保护作用,成为疫苗研究的靶点。本文综述O-抗原多糖的基因结构和合成、生物学功能及其在疫苗研制中的应用与前景。     

鼠伤寒沙门菌感染巨噬细胞铁稳态相关基因mRNA表达谱

用荧光定量PCR法检测鼠RAW264.7巨噬细胞感染与未感染鼠伤寒沙门菌后18种铁穗态相关基因的表达,评估宿主与病原体相互作用中铁稳态效应。研究显示,活的鼠伤寒沙门菌感染巨噬细胞1 h后可以诱导转铁蛋白受体表达,引起细胞内动态铁池相关基因的mRNA水平上长。基因表达分析显示,沙门菌通过诱导铁氧还原酶(Steap3)、铁膜转运蛋白(Dmt1)、铁调节因子Tfr2/Hfe以及铁调节蛋白(Irp1和Irp2)的表达主动吸收铁,而经铁转运蛋白(Fpn1)的铁外流并无明显改变。沙门菌在感染后1h积极地驱动了转铁蛋白介导的铁吸收程序。     

扁蓿豆MrLEA2基因的克隆和原核表达

从扁蓿豆(Medicago ruthenica L.)幼苗中克隆到一个编码晚期胚胎发生丰富蛋白的基因MrLEA2,Pfam数据库检索表明其编码产物属于LEA_2蛋白家族。半定量RT-PCR分析发现MrLEA2在幼苗中表达水平不受非生物胁迫(脱水、高盐和低温)和脱落酸诱导。利用MrLEA2基因构建原核表达载体,在大肠杆菌中实现了过量表达。通过斑点试验和菌落计数实验,对过量表达MrLEA2蛋白的大肠杆菌细胞在高盐(0.5mol/L NaCl和0.5mol/L KCl)、55℃高温和-20℃冷冻胁迫处理下的生长存活情况检测发现,MrLEA2蛋白过量表达能够明显提高大肠杆菌对上述胁迫的耐受性。研究表明,MrLEA2蛋白对高盐和温度胁迫引起的细胞损伤具有保护作用。     

沙门菌PhoP-PhoQ双组分信号转导系统

PhoP-PhoQ是调控沙门菌毒力的重要双组分信号转导系统,由组氨酸蛋白激酶PhoQ和反应调节蛋白PhoP组成。PhoP-PhoQ可调节沙门菌对Mg2+及其他周质环境的适应性,并调控沙门菌感染中毒力基因的转录和表达。PhoP-PhoQ调控的毒力基因参与沙门菌对上皮细胞的侵袭、胞内生存、对抗菌肽的抵抗反应、脂质A的修饰、Ⅲ型分泌系统效应蛋白的分泌等环节。PhoP-PhoQ还可与其他双组分信号转导系统或调节子合作,调控沙门菌的毒力。因此,PhoP-PhoQ双组分信号转导系统在沙门菌的毒力调控中发挥重要作用。     

携带HCV核心蛋白原核表达质粒与真核表达质粒的减毒伤寒沙门菌诱导小鼠免疫应答之比较

丙型肝炎病毒(HCV)核心蛋白是丙肝疫苗的重要候选抗原,然而,该蛋白因具有免疫调控作用而影响免疫应答的诱导。构建了HCV核心蛋白的两种表达质粒,一种是体内激活型原核表达质粒pZW-C,另一种是真核表达质粒pCI-C。将该两种质粒转化减毒鼠伤寒沙门菌SL7207,得到重组菌SL7207/pZW-C和SL7207/pCI-C,分别将重组菌口服接种小鼠,检测小鼠的免疫应答,结果发现:①SL7207/pCI-C免疫鼠的CD3 CD4 T细胞持续降低,而SL7207/pZW-C免疫鼠的CD3 CD4 T细胞无明显改变;②SL7207/pCI-C免疫只诱导低水平抗HCV核心蛋白抗体,加强免疫对抗体阳转率及抗体水平无明显影响,而SL7207/pZW-C免疫组所有小鼠均产生较高水平的抗核心蛋白抗体。③SL7207/pCI-C免疫鼠脾细胞的体外增殖活性、细胞毒性T细胞活性以及加强免疫对细胞免疫应答的增强作用均明显不及SL7207/pZW-C免疫鼠。结果提示:携带真核表达质粒pCI-C的沙门菌因在小鼠细胞内表达天然形式(结构以及磷酸化修饰)的HCV核心蛋白,可能通过对T细胞的免疫抑制作用而弱化免疫应答。而以携带原核表达质粒pZW-C的沙门菌免疫可避免这一问题,并具有接种方便,成本低廉等优点,从而可望作为基于HCV核心蛋白为靶抗原的HCV疫苗的候选免疫方式。     

肺炎克雷伯菌HdeA蛋白的抗酸理化性质鉴定

肺炎克雷伯菌是一种常见的院内条件致病菌,通常定植在人体的呼吸道和胃肠道。但肺炎克雷伯菌是怎样在胃液的强酸条件下存活并进入肠道尚不十分清楚。本研究发现,酸性条件能够诱导肺炎克雷伯菌KpHdeA基因表达上调。克隆KpHdeA基因并将其转化大肠杆菌,获得可溶性的KpHdeA蛋白并利用镍离子亲和层析纯化得到目的蛋白。光散射分析证明,酸性胁迫下,KpHdeA蛋白能够减少酸诱导的醇脱氢酶聚集。荧光实验证明,酸胁迫可以诱导KpHdeA蛋白的疏水基团暴露。圆二色谱实验证明,酸性条件能够诱导KpHdeA蛋白形成无序结构。此外KpHdeA蛋白的表达能提高大肠杆菌的耐酸能力。故推测,KpHdeA蛋白具有分子伴侣活性,并在肺炎克雷伯菌抵抗酸胁迫中起重要作用。     

植物热激蛋白的研究进展及其应用

热激蛋白是一组在进化上高度保守的蛋白质,是生物体受环境胁迫时产生应激反应,降低正常基因的表达,并启动热激基因而产生的一种特殊蛋白质,能使机体抵御不良的环境。本文综述了植物热激蛋白的种类、特点、诱导合成、细胞定位及其在生命科学研究中的应用。     

肠炎沙门菌肽脯氨酰顺反异构酶SlyD的基因敲除、表达及酶学特征

【目的】以肠炎沙门菌肽脯氨酰顺反异构酶SlyD为对象,构建基因缺失株及表达纯化该蛋白,为研究其在肠炎沙门菌致病性与应激等方面的作用奠定基础。【方法】参考Gen Bank登录的肠炎沙门菌基因组序列设计用于slyD基因敲除及原核表达的特异引物,运用自杀质粒介导的同源重组技术对肠炎沙门菌C50041 slyD基因进行敲除,构建C50041ΔslyD缺失株;原核表达SlyD蛋白,通过α-糜蛋白酶耦联法对其PPIase活性进行测定;利用生物信息学相关软件,分析SlyD蛋白的氨基酸序列及功能域。【结果】PCR鉴定与测序结果证明成功构建了肠炎沙门菌C50041ΔslyD缺失株,其生长特性与野生株基本一致;SDS-PAGE及PPIase活性分析表明,获得了具有生物活性的可溶性SlyD蛋白;生物信息学分析显示SlyD蛋白由FKBP样肽脯氨酰顺反异构酶结构域、分子伴侣功能域和金属结合区域3个功能区域组成。【结论】成功获得了肠炎沙门菌C50041ΔslyD缺失株和具有PPIase活性的重组SlyD蛋白。     

Oxidation-reduction potential regulates RpoS levels in Salmonella Typhimurium

AIMS: The aim of this work was to investigate the connection between oxidation-reduction (redox) potential and stationary phase induction of RpoS in Salmonella Typhimurium. METHODS AND RESULTS: A lux-based reporter was used to evaluate RpoS activity in S. Typhimurium pure cultures. During growth of S. Typhimurium, a drop in the redox potential of the growth medium occurred at the same time as RpoS induction and entry into stationary phase. An artificially induced decrease in redox potential earlier during growth reduced the time to RpoS induction and Salmonella entered the stationary phase prematurely. In contrast, under high redox conditions, Salmonella grew unaffected and entered the stationary growth phase as normal, although RpoS induction did not occur. As a consequence, stationary phase cells grown in the high redox environment were significantly more heat sensitive (P < 0.05) than those grown under normal conditions. CONCLUSIONS: This work suggests that redox potential can regulate RpoS levels in S. Typhimurium and can thus, control the expression of genes responsible for thermal resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to manipulate RpoS induction and control stationary phase gene expression can have important implications in food safety. Early RpoS induction under low redox potential conditions can lead to enhanced resistance in low cell concentrations to inimical processes such as heat stress. Inhibition of RpoS induction would abolish stationary phase protective properties making cells more sensitive to common food control measures.     

中华蜜蜂ZFP37基因的生物信息学分析及高温下表达特性的研究

锌指蛋白（Zinc finger proteins,ZFPs）是一类在真核生物体内广泛分布的蛋白质。锌指蛋白作为一类转录因子,它能够调控基因的表达和细胞的分化,最近的研究显示其在动植物抗逆方面也发挥着重要作用。本研究对中华蜜蜂 Apis cerana cerana ZFP37的蛋白结构进行了预测分析,并通过qRT-PCR分析了中华蜜蜂在遭受高温胁迫时ZFP37的表达情况,进一步了解锌指蛋白在中华蜜蜂应对热胁迫过程中的作用。结果显示,中华蜜蜂ZFP37可编码123个氨基酸,蛋白分子量为13.7 kDa,无跨膜结构。氨基酸同源序列比对结果表明,中华蜜蜂ZFP37序列与蜜蜂科昆虫的相似性最高,与其他膜翅目昆虫的相似性存在差异。基因的表达模式显示,ZFP37在高温下表达升高,此外,胁迫时间的增加也可导致ZFP37表达的升高。这些结果表明ZFP37对于中华蜜蜂应对热应激有重要的生物学意义。     

Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42°C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.     

Regulatory interfaces between the stress protein response and other gene expression programs in the cell

The stress protein response involves the immediate reprogramming of gene expression in cells exposed to proteomic insult leading to massive synthesis of heat shock proteins (HSP). We have examined the outcome when cells are induced to activate two other gene expression programs--the acute inflammatory response and entry of quiescent cells into the cell cycle--and then exposed to protein stress. We find that these responses are mutually antagonistic with, on the one hand, heat shock factor 1 (HSF1) inhibition through the phosphorylation of inhibitory serine residues after inflammatory or mitogenic stimulus and, on the other hand, after stress, HSF1 directly repressing the promoters of genes that mediate acute inflammation and mitogenesis. The expression of the stress protein response during periods of acute protein damage was shown to lead to efficient activation of HSF1 and HSP expression accompanied by repression of other gene expression programs.     

Requirement of Fur for the full induction of Dps expression in Salmonella enterica serovar typhimurium

The Dps protein, which is overexpressed in harsh environments, is known to play a critical role in the protection of DNA against oxidative stresses. In this study, the roles of Fur in the expression of the dps gene in Salmonella and the protection mechanisms against oxidative stress in Salmonella cells preexposed to iron-stress were investigated. Two putative Fur boxes were predicted within the promoter region o f th e S. typhimurium dps gene . The profile of dps expression performed by the LacZ reporter assay revealed growth-phase dependency regardless of iron-status under the culture conditions. Thefur mutant, chi4659, evidenced a reduced level of beta-galactosidase as compared to the wild-type strain. The results observed after the measurement of the Dps protein in various Salmonella regulatory mutants were consistent with the results acquired in the reporter assay. This evidence suggested that Fur performs a function as a subsidiary regulator in the expression of dps. The survival ability of Salmonella strains after exposure to oxidative stress demonstrated that the Dps protein performs a pivotal function in the survival of stationary-phase S. typhimurium against oxidative stress. Salmonella cells grown in iron-restricted condition required Dps for full protection against oxidative stress. The CK24 (Deltadps) cells grown in iron-replete condition survived at a rate similar to that observed in the wild-type strain, thereby suggesting the induction of an unknown protection mechanism(s) other than Dps in this condition.     

Reduced Enzyme Activity Following Hsp70 Overexpression in Drosophila melanogaster

Acclimation to environmental change can impose costs to organisms. One potential cost is the change in cell metabolism that follows a physiological response, e.g., high expression of heat shock proteins may alter specific activity of important enzymes. We examined the significance of this cost in a pair of Drosophila melanogaster lines transformed with additional copies of a gene that encodes the heat shock protein, Hsp70. Heat shock induces Hsp70 expression in all lines, but lines with extra copies produce much more Hsp70 than do excision control strains. The consequence of this supranormal Hsp70 expression is to reduce specific activity of both enzymes analyzed, adult alcohol dehydrogenase (ADH), which is heat sensitive, and lactate dehydrogenase, which is not. Strain differences were most pronounced under those conditions where Hsp70 expression was maximized, and not where the heat stress denatured proteins. That result supported the idea that Hsp70 expression is constrained evolutionarily by its tendency to bind nascent peptides when overabundant within the cell.     

Acute Heat Stress Changes Protein Expression in the Testes of a Broiler-Type Strain of Taiwan Country Chickens

Heat stress leads to decreased fertility in roosters. This study investigated the global protein expression in response to acute heat stress in the testes of a broiler-type strain of Taiwan country chickens (TCCs). Twelve 45-week-old roosters were randomly allocated to the control group maintained at 25°C, and three groups subjected to acute heat stress at 38°C for 4 h, with 0, 2, and 6 h of recovery, respectively. Testis samples were collected for hematoxylin and eosin staining, apoptosis assay, and protein analysis. The results revealed 101 protein spots that differed significantly from the control following exposure to acute heat stress. The proteins that were differentially expressed participated mainly in protein metabolism and other metabolic processes, responses to stimuli, apoptosis, cellular organization, and spermatogenesis. Proteins that negatively regulate apoptosis were downregulated and proteins involved in autophagy and major heat shock proteins (HSP90α, HSPA5, and HSPA8) were upregulated in the testes of heat-stressed chickens. In conclusion, acute heat stress causes a change in protein expression in the testes of broiler-type B strain TCCs and may thus impair cell morphology, spermatogenesis, and apoptosis. The expression of heat shock proteins increased to attenuate the testicular injury induced by acute heat stress.