Effects of <Emphasis Type="Italic">TFAR19</Emphasis> gene on the <Emphasis Type="Italic">in vivo</Emphasis> biorheological properties and pathogenicity of mouse erythroleukemia cell line MEL

After injecting VP16, MEL cells and MEL-TF19 cells into the body of mice, with those injected with the same dose of saline as the control group, we observed the mice for their blood pictures, histological changes of the liver and spleen, and the hemorhelogical indexes within 4 weeks. The results indicated that after injecting MEL cells, the mice entered into a pathological status similar to erythroleukemia, which had the following exhibitions: the tissue structures of the liver and spleen were damaged, a mass of proerythroblasts, basophil erythroblasts and polychromatophilic erythroblasts could be observed on the smears of the bone marrow and spleen, and the deformability and orientation ability of erythrocytes were both depressed. The pathogenicity of MEL-TF19 cells carrying TFAR19 gene was obviously lower than that of MEL cells, and the MEL-TF19 cells even lost their faintish pathogenicity under the apoptosis-inducing effect of the chemotherapeutic reagent. The outcome from the animal experiments suggests that the TFAR19 gene suppresses the pathogenicity of MEL cells to the mice, and the effect may be better exerted with the synergy of the chemotherapeutic reagent.     

Effects of TFAR19 gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL

Using the method of gene transfection with liposome, we obtained the mouse erythroleukemia cell line MEL-TF19, which stably carries TFAR19, a novel apoptosis-related gene. The expression of TFAR19 was detected by Western blot. Growth curve and flow cytometry analysis showed that after being transfected with TFAR19 gene, the growth of MEL-TF19 is suppressed and its apoptosis is accelerated because of the serum deprivation. Our biorheological study indicated that in the apoptotic process, compared with MEL cells, MEL-TF19 cells exhibit larger osmotic fragility, lower cell surface charge density, increased elastic modulus K ₁ which is inversely proportional to cells’ maximal deformation ability, obviously diminished surface viscosity μ, with elastic modulus K ₂having no distinct changes. The above results provided some bases for recognizing the function of TFAR19 completely from the viewpoint of biorheology.     

Effects of TFAR19 gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL

Apoptosis is a genetically controlled program of cellular self-destruction, which plays a key role in the development and homeostasis of virtually all animals. In recent years, Liu et al.[1] found a novel apoptosis-related gene TFAR19 (TF-1 cells apoptosis-related gene 19). It is conserved in evolution and distributed extensively in tissues. The related study showed that its coding protein is probably very important for the signal transduction in cell nucleus, regulating the cell proliferat…     

Effects of <Emphasis Type="Italic">TFAR19</Emphasis> gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL

Using the method of gene transfection with liposome, we obtained the mouse erythroleukemia cell line MEL-TF19, which stably carries TFAR19, a novel apoptosis-related gene. The expression of TFAR19 was detected by Western blot. Growth curve and flow cytometry analysis showed that after being transfected with TFAR19 gene, the growth of MEL-TF19 is suppressed and its apoptosis is accelerated because of the serum deprivation. Our biorheological study indicated that in the apoptotic process, compared with MEL cells, MEL-TF19 cells exhibit larger osmotic fragility, lower cell surface charge density, increased elastic modulus K1 which is inversely proportional to cells' maximal deformation ability, obviouslydiminished surface viscosity μ, with elastic modulus K2 having no distinct changes. The above results provided some bases for recognizing the function of TFAR19 completely from the viewpoint of biorheology.     

加州鲈源鰤鱼诺卡氏菌的分离鉴定及致病性

[背景] 鰤鱼诺卡氏菌（Nocardia seriolae）是一种严重危害水产养殖业的病原菌,可引起以体表溃疡、出血及组织器官形成结节为特征的鱼类慢性肉芽肿疾病,目前尚无有效的防治方法。[目的] 明确引起安徽省临泉县某养殖场加州鲈（Micropterus salmonoides）结节病的病原菌,探讨其致病性,为该病的有效防治提供科学依据。[方法] 取肝脏结节病灶接种于TSB培养基分离优势细菌,利用表型检查结合分子生物学方法鉴定分离菌株。进一步通过检测分离菌株的毒力基因、测定其对加州鲈的半数致死量（LD₅₀）以及所感染加州鲈的组织病理学变化与组织载菌量,分析其致病性。[结果] 从病鱼体内分离到一株优势菌株NI,综合NI分离株的表型特性、16S rRNA基因序列与鰤鱼诺卡氏菌参考株相应序列的一致性以及特异性PCR扩增结果,确定其为鰤鱼诺卡氏菌。鰤鱼诺卡氏菌NI分离株携带毒力基因gapA、ibeA和mip,人工回归感染后加州鲈出现与自然病例相似的症状,其对加州鲈的LD₅₀为2.58×10⁶ CFU/尾。组织病理学观察到头肾、心脏、肝脏、胃和脾脏均出现慢性肉芽肿病变,肠管肌层疏松、肠绒毛脱落,肌肉组织中肌纤维疏松、间隙增宽。qPCR检测结果显示,组织中鰤鱼诺卡氏菌载量由高到低依次为头肾、心、肝、胃、脾、肠和肌肉。[结论] 鰤鱼诺卡氏菌是引起此次加州鲈结节病的病原菌,对该菌致病性的研究为加州鲈诺卡氏菌病的防控提供了理论依据。     

TFAR19 gene changes the biophysical properties of murine erythroleukemia cells

TFAR19 is a novel apoptosis-related gene and can accelerate cell apoptosis in the presence of apoptosis inducements. Here, we studied the effects of TFAR19 on some biophysical properties of mouse erythroleukemia (MEL) cells and their molecular and structural basis. After transfected with TFAR19 and apoptosis inducement, MEL revealed a high cell membrane fluidity, a decrease in resynthesis of phospholipids, an increase in the proteins/nucleic acids ratio, a relatively orderly cytoskeleton network, an impaired deformability, a low integrin aM expression, and a decrease in adhesion to endothelial cells. These findings suggest the potential of TFAR19 for antitumor cell migration, and thus for antitumor gene therapy.     

The in vivo distribution of Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

蛋白-O-岩藻糖基转移酶1基因CfPOFUT1在果生炭疽菌中的功能分析

【目的】蛋白-O-岩藻糖基转移酶1 (protein O-fucosyltransferase 1,POFUT1)是催化蛋白质O-岩藻糖基化的关键酶,在动物和人体内被证明调控一系列的生理病理过程,然而POFUT1基因在果生炭疽菌乃至真菌中还未见报道。本研究旨在克隆果生炭疽菌中CfPOFUT1基因,并分析其生物学功能。【方法】利用RT-PCR技术扩增CfPOFUT1的基因并进行生物信息学分析,构建了CfPOFUT1基因的沉默和过表达载体,通过PEG介导法将载体导入原生质体中获得CfPOFUT1基因的沉默和过表达突变体。测定了野生型菌株、CfPOFUT1沉默菌株和过表达菌株在PDA上的菌丝生长、分生孢子产生、萌发与附着胞形成、胁迫应答和致病力、杀菌剂敏感性等生物学表型。【结果】与野生型菌株相比,基因过表达突变体产孢量显著增加,致病力增强,对嘧菌酯敏感性降低,但对多菌灵和咪鲜胺敏感性增强。基因沉默突变体产孢量减少,细胞壁完整性、内质网应激敏感性提高,致病力减弱,对嘧菌酯敏感性提高,但对多菌灵和咪鲜胺敏感性降低。【结论】CfPOFUT1基因参与调控果生炭疽菌分生孢子产量,细胞壁完整性、内质网对应激和药剂敏感性,并对其致病性也具有一定的影响。     

高致病性美人鱼发光杆菌美人鱼亚种胞外产物的毒力特性分析

【目的】美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp. damselae,PDD)是一种可导致多种海洋生物患病的重要病原菌。本研究以从我国海水养殖环境中分离的具有强磷脂酶活性、强溶血性表型且具有高致病性的2株PDD菌株为研究对象,分析PDD菌株胞外产物(extracellularproducts,ECP)的致病性及细胞毒性,及其与菌株致病性表型的相关性。【方法】利用培养基平板法测定菌株PDD1605、PDD1608的ECP体外磷脂酶活性和溶血性;通过人工感染实验测定PDD1605和PDD1608活菌株及其ECP对许氏平鲉的致病性,并进行组织病理切片观察组织的病理损伤;通过向人胚肾细胞系HEK293T和小鼠成纤维细胞系MCFS等2种培养细胞中添加菌株PDD1605和PDD1608的ECP测定其对哺乳动物的细胞毒性。【结果】人工感染PDD1605和PDD1608活菌株对许氏平鲉均表现为高致病性,5×108CFU/mL的菌液浓度24h内受试鱼全部死亡;菌株PDD1605和PDD1608的ECP对许氏平鲉同样表现为高致病性,受试鱼的死亡曲线与感染活菌株的...     

一株牙鲆源黏质沙雷氏菌YP1的分离鉴定及致病性分析

黏质沙雷氏菌(Serratia marcescens)是引起人类、动物及植物感染的重要条件致病菌,但其作为鱼类致病菌却鲜有报道。【目的】本研究以从患病牙鲆(Paralichthys olivaceus)病灶处分离的一株黏质沙雷氏菌YP1为研究对象,分析黏质沙雷氏菌对鱼类的致病性及对疾控的影响。【方法】利用形态学、分子生物学及生理生化实验综合鉴定菌株YP1;利用菌株YP1进行人工感染实验、组织病理实验及药敏试验,研究其感染症状、组织病理学、毒力和药物敏感性。【结果】分离自患病牙鲆体表溃疡病灶处的菌株YP1鉴定为黏质沙雷氏菌。感染实验结果显示,牙鲆和斑马鱼的半数致死量(LD₅₀)分别为3.44×10⁷CFU/g和6.28×10⁵CFU/g,除牙鲆外菌株YP1对其他鱼类也具有高致病性;菌株YP1主要导致牙鲆腹水,同时伴有呼吸急促、摄食减弱、脱肛、白便、鳃缺血及多脏器膨大出血等症状,并随着感染时间的延长对脏器损伤呈加重趋势。病理组织切片结果显示,菌株YP1对牙鲆鳃、肠、肝、脾、肾、心均造成损伤。药敏试验结果表明,YP1对左氧氟沙星、诺氟沙星等14种药物敏感;但对氨苄西林、头孢拉定等19种药物具有耐药性。【结论】本研究结果证实了黏质沙雷氏菌是能导致牙鲆腹水病的一种病原菌,同时对其他鱼类也具高致病性,为该菌感染鱼类导致疾病的检测、鉴别和防治提供科学依据。     

大熊猫源肺炎克雷伯菌生物学特性

【背景】肺炎克雷伯菌是仅次于大肠杆菌的常见条件致病菌之一,严重时可导致大熊猫发生出血性肠炎、全身性败血症等。【目的】明确大熊猫源肺炎克雷伯菌的生物学特性,对防控该病作出科学指导。【方法】分别采用结晶紫染色法、拉丝实验、K-B纸片法和PCR技术对46株大熊猫源肺炎克雷伯菌的生物被膜形成能力、高黏性表型、耐药表型和15种常见毒力基因等生物学特性进行研究,并根据以上生物学特性选择一株可能具有致病性的分离菌pneumoniae-X-5,研究其对小鼠的致病性。【结果】46株肺炎克雷伯菌均可形成荚膜;12株为高黏性表型肺炎克雷伯菌;能形成生物被膜的菌株占比为65%(30/46);分离出的46株菌中多重耐药菌株占58%(27/46),对氨苄西林、苯唑西林、青霉素、万古霉素呈100%耐药;毒力基因检出率最高的为ureA(91.30%,42/46)。pneumoniae-X-5菌株对小鼠的LD₅₀为8.9×10⁴CFU/mL;该菌株攻毒小鼠肺泡间隔增厚,炎性细胞浸润,肝细胞变性坏死,脾充血,十二指肠黏膜上皮和固有层分离,固有层部分细胞坏死。死亡小鼠脾脏含细菌量最多,其次为肝脏。【结论】本试验阐明了部分大熊猫源肺炎克雷伯菌的多重耐药性、能形成生物被膜、具有高黏表型等病原生物学特性,为大熊猫肺炎克雷伯杆菌病的防控及临床治疗提供了科学依据。     

鳜源致病性舒伯特气单胞菌的分离鉴定及药敏试验

【背景】舒伯特气单胞菌(Aeromonas schubertii)广泛分布于淡、海水水体和底泥中,致病株已在我国养殖鳢科鱼类中流行,也感染其他经济鱼类,导致暴发性死亡。【目的】对病鳜(Siniperca chuatsi)的病原进行鉴定,确定分离菌的致病性及药物敏感性,为该病临床治疗提供参考。【方法】采集病鳜脾肾组织进行PCR或RT-PCR扩增其常见病毒,采集病鳜肝脏和腹水分离培养细菌,PCR扩增代表菌株的gyrB、16S rRNA和毒力基因,鉴定其生理生化特征,并进行药物敏感性试验和人工感染试验。【结果】病鳜的传染性脾肾坏死病毒、鳜蛙病毒、鳜弹状病毒检测结果为阴性,肝脏和腹水均存在大量细菌;代表菌株Gui210820被鉴定为舒伯特气单胞菌,携带溶血素、气溶素、弹性蛋白酶和磷脂酶毒力基因,腹腔注射感染鳜的半数致死浓度(LD50)为3.16×105 CFU/mL;菌株Gui210820对四环素、卡那霉素、复方新诺明等6种抗菌药物耐药,对强力霉素中介,对恩诺沙星、新霉素、氟苯尼考等11种抗菌药物敏感。【结论】本试验从病鳜组织分离到致病性舒伯特气单胞菌,水产准许用药物恩诺沙星、新霉素、氟苯尼考...     

Trypanosoma brucei brucei: A comparison of gene expression in the liver and spleen of infected mice utilizing cDNA microarray technology

Trypanosoma brucei brucei, the infectious agent of the disease known as Nagana, is a pathogenic trypanosome occurring in Africa, where it causes significant economic loss to domesticated livestock. Although many studies on the histopathology of organs of mice infected with T. b. brucei have been reported, little work has been done regarding gene expression in these organs in infected mice. In this paper, we describe the use of cDNA microarray to determine gene expression profiles in the liver and spleen of mice infected with T. b. brucei (STIB 920) at peak parasitaemia (12 days after infection). Our results showed that a total of 123 genes in the liver and 389 genes in the spleen were expressed differentially in T. b. brucei infected mice. In contrast, however, in an acute infection in mice caused by Trypanosoma brucei evansi, a species genetically related to T. b. brucei, 336 genes in the liver and 190 genes in the spleen were expressed, differentially, indicating that the liver of mice was more affected by the acute T. b. evansi infection whilst the spleen was more affected by the subacute T. b. brucei infection. Our results provide a number of possible reasons why mice infected with T. b. evansi die sooner than those infected with T. b. brucei: (1) mice infected with T. b. evansi may need more stress response proteins to help them pass through the infection and these are probably excessively consumed; (2) proliferating cell nuclear antigen was more down-regulated in the liver of mice infected with T. b. evansi, which indicated that the inhibition of proliferation of hepatocytes in mice infected with T. b. evansi might be more severe than that in T. b. brucei infection; and (3) more hepatocyte apoptosis occurred in the mice infected with T. b. evansi and this might be probably the most important reason why mice died sooner than those infected with T. b. brucei. Studies of the changes in the gene expression profile in the liver and spleen of mice infected with T. b. brucei may be helpful in understanding the mechanisms of pathogenesis in Nagana disease at the molecular level. By comparing the gene profiles of the liver and spleen of mice infected with T. b. brucei with T. b. evansi, we have identified a number of factors that could explain the differences in pathogenesis in mice infected with these two African trypanosomes.     

cDNA cloning and expression of an apoptosis-related gene, humanTFAR15 gene

By means of cDNA-RDA method, some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these fragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1 218 nucleotides and encodes 212 amino acids. The putative protein product of TFAR15 is partially homologous toC. elegans protein C14A4.11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung, and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-1 cells after GM-CSF withdrawal.In vitro analysis showed that the recombinant TFAR15 protein could inhibit the natural cell death of 293 cells, an embryonic kidney cell line.     

Molecular cloning of protease gene(s) from Xanthomanas campestris pv. campestris: Expression in Escherichia coli and role in pathogenicity

Summary The recombinant plasmid pIJ3070 isolated from a genomic library of Xanthomonas campestris pv. campestris constructed in the conjugal cosmid pLAFR3 contains protease gene(s) which can be expressed in Escherichia coli. Tn5 mutagenesis and subcloning revealed that the protease structural gene(s) is(are) located in a ca. 10 kb EcoRI fragment. Several protease-minus mutants of X. c. campestris were obtained by Tn5 mutagenesis of pIJ3070 and marker exchange techniques. Studies of pathogenicity of these Tn5 mutants showed that the protease is not critically important for the pathogenicity of X. c. campestris on turnip plants but may play a minor role in disease development.Abbreviations Gm gentamicin - Km kanamycin - Rif rifampicin - Spc spectinomycin - Sm streptomycin - Tc tetracycline     

沙门菌噬菌体抗性菌的筛选鉴定及致病力研究

【目的】筛选鉴定沙门菌噬菌体侵染裂解过程中的抗性菌株,研究抗性菌株的生物学特性及致病力的差异,为解决噬菌体治疗应用中的抗性菌问题提供理论依据。【方法】本研究通过次级感染法和双层平板法筛选沙门菌噬菌体抗性菌,通过生物学特性和毒力基因检测比较宿主菌ATCC 13076及其噬菌体抗性菌株R3之间的差异,并通过小鼠攻毒实验和细胞粘附实验比较致病力强弱。【结果】噬菌体抗性菌株R3的生长速度较宿主菌略慢;生化及毒力基因检测均表明抗性菌株与宿主菌无差异;与宿主菌相比,抗性菌R3的LD50增加了74.8%(P>0.05);对MODE-K细胞粘附能力稍弱,但是差异不显著。【结论】该研究表明,与噬菌体宿主菌相比,噬菌体抗性菌株的生物学特性和毒力基因并没有改变,对小鼠致病力减弱,但是对MODE-K细胞粘附能力差异不显著。     

毒力基因yqeH功能分析及对禽致病性大肠杆菌致病性的影响

【背景】禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)可引起禽类急性或亚急性感染,在近年新发现的大肠杆菌Ⅲ型分泌系统2 (Escherichia coli type III secretion system 2,ETT2)中,毒力基因yqeH对其致病性的影响尚不明确。【目的】探究yqeH在APEC致病过程中的作用,为后期深入研究ETT2致病机制奠定基础。【方法】利用Red同源重组技术构建yqeH缺失株ΔyqeH及其回复株CΔyqeH,通过运动性、生物被膜形成能力、抗逆性、抗血清杀菌能力等试验分析yqeH对APEC生物学功能的影响,并通过细胞黏附、侵袭试验、致病力测定及荧光定量PCR检测细胞炎性因子转录水平,探究yqeH对APEC感染宿主的影响。【结果】构建了缺失株ΔyqeH和回复株CΔyqeH;生物学特性试验结果表明,与野生株APEC81相比,缺失株ΔyqeH生物被膜形成能力、运动能力降低,对酸、碱、渗透压、氧化休克的耐受力降低,抗血清杀菌能力及致病力显著降低;与野生株APEC81相比,缺失株ΔyqeH对鸡气管黏膜上皮细胞的黏附及侵袭能...     

苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能

【目的】研究表明,细胞色素P450(CYP)在死体营养型真菌的毒素合成代谢中发挥重要作用,预测可能与病原菌致病相关。论文对苹果树腐烂病菌(Valsa mali)毒素合成基因簇中的1个上调表达的CYP基因Vmcyp5进行生物学功能研究,明确CYP基因对病原菌致病力影响,为细胞色素P450基因家族对苹果树腐烂病菌致病机理的进一步研究提供依据。【方法】通过Double-joint PCR和PEG介导的原生质体转化技术获得具有G418抗性的突变体,并对突变体进行PCR检测及Southern blotting验证得到单拷贝敲除突变体。将目的基因片段重新导入敲除突变体,筛选获得互补突变体。最终对野生型菌株及敲除突变体、互补突变体进行菌落、产孢及致病力观察,利用SPSS软件对数据进行差异显著性分析,并利用q RT-PCR技术分析突变体黑色素基因簇的表达水平。【结果】通过基因敲除技术获得1个Vmcyp5基因的敲除突变体。与野生型菌株相比,Vmcyp5基因的敲除突变体菌落呈白色,产孢量减少51.3%。q RT-PCR分析发现敲除突变体黑色素基因簇基因表达量降低。重要的是,敲除突变体致病力较野生型菌株降低24.5%。互补突变体菌落颜色、产孢及致病力近似恢复至野生型菌株水平。【结论】Vmcyp5基因与病原菌黑色素合成、子实体的产生和致病力相关。     

表达新冠病毒S基因重组狂犬病病毒的构建及其免疫原性

【背景】新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)在全球流行已近3年,除对人类造成了巨大伤害,也影响了人类的伴侣动物。人的COVID-19疫苗已在全球应用,但动物用的新冠病毒疫苗却鲜有报道。【目的】研制兽用新冠病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)和狂犬病病毒(rabies virus,RABV)的二联苗。【方法】将合成的SARS-CoV-2 S基因和S1基因分别克隆至RABV弱毒疫苗株rHEP-Flury基因组G与L基因间,并将2个重组质粒分别与辅助质粒共转染至BHK-21细胞中,拯救重组病毒rHEP-nCOV-S和rHEP-nCOV-S1。通过RT-PCR、Western blotting和荧光抗体染色,验证重组病毒、确证S和S1蛋白在RABV中成功表达。再将重组病毒接种NA细胞及成年小白鼠,测定病毒的体外生长特性、重组病毒的致病性及免疫原性。【结果】免疫荧光结果显示,转染7d后细胞上清均出现了绿色免疫荧光,表明已成功拯救嵌合SARS-CoV-2S和S1基因的重组病毒RABV rHEP-nCOV-S和rHEP-nCOV-S1,并且rHEP-nCOV-S1的增殖和扩散能力强于亲本株rHEP-Flury,但rHEP-nCOV-S与亲本株无显著差异。Western blotting结果显示,在目的位置处均出现72kDa和144kDa特异性条带,表明S和S1蛋白在重组RABV中高效表达。重组病毒免疫6周KM小鼠后,小鼠的体重变化与亲本RABV基本一致,重组病毒诱导小鼠产生狂犬中和抗体。【结论】本研究拯救出了嵌合SARS-CoV-2 S/S1基因的重组RABV,为动物COVID-19载体疫苗的研发奠定了基础。