Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.     

水稻单C_2H_2型锌指基因ZOS2-01的表达与功能分析

拟南芥超雄基因（SUPERMAN,SUP）是一个单C2H2锌指基因。该基因突变后会造成雌蕊同源转化为雄蕊,正常的心皮发育受阻,因此SUP在控制花第3/4轮边界的建立和胚珠的发育中起重要作用。为了探知水稻中的SUP同源基因是否也存在类似的功能,我们根据拟南芥SUP的功能结构域,从水稻中克隆了一个与SUP类似基因,命名为ZOS2-01,并对其表达和功能进行了分析。结果表明,ZOS2-01与拟南芥的SUP和矮牵牛的PhSUP1在系统进化树上处于同一分支且与SUP的功能结构域完全相同。定量PCR和GUS表达分析表明,除胚乳外,ZOS2-01几乎在所有组织和器官中表达,但在幼穗中的表达量最高,暗示它可能参与了水稻的营养生长和花器官发育的调节。然而,采用RNA干扰和过量表达的方法减少和增加ZOS2-01的表达并没有明显影响水稻的生长发育,转基因水稻的营养生长正常,花器官发育也没有明显异常且结实正常。这些结果表明,尽管水稻ZOS2-01与拟南芥SUP具有相同的功能结构域,但它们的表达和功能可能已出现了分化,或者水稻中存在多个功能冗余的SUP类似基因。     

根癌农杆菌Ⅵ型分泌系统关键组分ClpⅤ型ATPase基因表达及活性位点分析

细菌Ⅵ型分泌系统（type Ⅵ secretion system,T6SS）是近年发现的一种分布广泛且与细菌致病性密切相关的新型分泌系统,ClpⅤ型ATPase是不同细菌T6SS中均含有的关键保守组分,据推测可能为底物蛋白的分泌提供动力,但对其生化功能尚未进行深入鉴定。在本研究中,我们克隆了根癌农杆菌（Agrobacterium tumefaciens）C58中的ClpⅤ型ATPase基因atu4344并在大肠杆菌中表达,酶学检测证实了其ATPase活性,定点突变分析发现K232/608和E299/675突变体均丧失ATPase活性,表明Walker A模体和Walker B模体与其活性密切相关。这是首次对植物病原细菌T6SS中的ClpⅤ型ATPase进行生化鉴定和突变分析研究。     

成团泛菌低分子脂多糖的急性毒性、刺激性、致敏性及遗传毒性评估

本研究对成团泛菌低分子脂多糖（Pantoea agglomerans lipopolysaccharide,LPSp）的安全性进行初步评估。本研究采用一次限量法,用昆明种小鼠进行LPSp急性经口毒性试验,了解LPSp的急性毒性;采用新西兰兔分别进行LPSp急性和多次皮肤刺激性试验以及急性眼刺激性试验,了解LPSp的皮肤和粘膜刺激性;采用豚鼠进行LPSp皮肤变态反应试验,了解LPSp的致敏性;应用平板掺入法进行鼠伤寒沙门氏菌/回复突变试验和小鼠骨髓细胞微核试验考察LPSp的遗传危害。急性毒性试验结果显示,LPSp对小鼠经口一次灌胃的LD50大于5000mg/kg体重,属实际无毒级别;LPSp急性和多次皮肤刺激性试验以及急性眼刺激性试验结果显示,皮肤刺激和眼刺激积分均为0分,LPSp对皮肤无刺激性、对眼睛无急性刺激性;在皮肤变态反应试验中,LPSp在各观察时间点的皮肤变态反应积分均为0分,其致敏率均为0%,说明LPSp对豚鼠无致敏性;LPSp的鼠伤寒沙门氏菌/回复突变试验结果呈阴性（P〉0.05）;LPSp的小鼠骨髓细胞微核试验结果亦呈阴性,LPSp各剂量组的微核发生率与阴性对照组未见统计学差异（P〉0.05）,而与阳性对照组有明显差异（P〈0.01）。本研究结果表明,在本实验剂量范围内,LPSp对小鼠经口毒性极低,属实际无毒级别,对家兔皮肤和眼睛无明显刺激性,对豚鼠无致敏性,对所试菌株和小鼠体细胞无诱变性和致突变性。     

Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays.     

猴痘病毒PCR检测方法的建立

从GenBank上调取猴痘病毒的基因序列,经过分析,找出了猴痘病毒的特异性靶基因序列F3L片段,人工合成猴痘病毒MPV(AF380138)F3L基因片段(48048-48509bp)并插入质粒,作为病毒检测的模拟阳性模板。根据该序列设计并合成PCR引物,对模拟的阳性模板进行扩增,结果能扩增出与目的片段大小一致的条带。建立了PCR检测方法,经条件优化后,对鸡痘、禽痘、羊痘等14种相似病毒核酸进行PCR扩增,发现具有良好的特异性。PCR方法能扩增出0.3pg的阳性模板,显示建立的PCR方法具有高效、快速、特异、灵敏的特点,可用于口岸猴痘病毒的检疫。     

猫瘟热病毒胶体金快速检测试纸的研究

利用双抗体夹心和胶体金免疫层析技术的原理,在玻璃纤维膜、硝酸纤维膜的检测线(T)和对照线(C)上分别喷上胶体金与猫瘟热病毒(FPV)单克隆抗体(3C4)的偶联物、FPV多克隆抗体和羊抗鼠IgG单克隆抗体,制成检测FPV抗原的猫瘟热病毒胶体金快速检测试纸,对临床26份虎粪便样本进行检测,并与PCR检测法及韩国(ASAN ...     

结缕草根际联合固氮菌的分离及初步鉴定

联合固氮菌在植物根际土壤生长或定植于植物根表细胞,这类细菌可将空气或土壤中的氮素转化为氨态氮供植物直接吸收利用.结合气相色谱仪,利用乙炔还原等方法对结缕草根际联合固氮菌进行了分离和鉴定.结果表明:从结缕草根际土壤中分离获得9株联合固氮菌株,菌株的固氮酶活性相差较大,为67.5～343.7 C2H4nmol/(ml·h)...