伯氏疏螺旋体套式PCR检测方法建立及应用

为了解新疆北疆地区硬蜱携带莱姆病病原伯氏疏螺旋体的流行现状,选择莱姆病病原伯氏疏螺旋体5S-23S rRNA基因间隔区设计嵌套式引物,建立了具有较高敏感性和特异性的伯氏疏螺旋体套式PCR检测方法,应用该方法对2018—2019年在新疆北疆6个地区采集的硬蜱进行伯氏疏螺旋体带菌率检测。结果显示:伯氏疏螺旋体总阳性率为15.17%(81/534),其中青河县阳性率最高,达23.64%(26/110)。提示:应进一步加强及重视北疆地区莱姆病的防控工作。     

养狗要防莱姆病

莱姆病是由伯氏疏螺旋体引起的一种蜱媒螺旋体病。目前,已有19个国家发现该病流行。我国在山东、新疆、安徽、黑龙江等省区也发现了莱姆病患者。 以前认为蜱虫是莱姆病的主要传播者,近来,临床研究人员又发现狗和蝇类也能携带伯氏疏螺旋体而传播该病,而且狗感染伯氏螺旋体的的危险性更大。当携带伯氏螺旋体的蜱叮咬人时,寄生在蜱体内     

伯氏疏螺旋体分型技术研究进展

伯氏疏螺旋体是莱姆病的病原体,伯氏疏螺旋体基因型与临床表现、地理环境、媒介物种、宿主动物等有密切关系。目前伯氏疏螺旋体分型方法主要有血清学分型和分子生物学分型。本文主要对伯氏疏螺旋体的国内外分型技术进行了综述。     

新疆北疆部分地区鼠类伯氏疏螺旋体携带情况调查

为了解新疆北疆地区鼠类伯氏疏螺旋体携带情况及其基因型分布特征,2021年1—9月在新疆昌吉州吉木萨尔县、伊犁哈萨克自治州新源县、塔城地区乌苏市巴音沟,采集147只野生鼠类的肾脏和膀胱组织样本,采用实时荧光定量PCR和巢式PCR方法,检测鼠类中的伯氏疏螺旋体携带情况;以伯氏疏螺旋体5S—23S基因间隔区进行巢式 PCR检测,对检出的阳性样本进行基因测序及序列分析,确定伯氏疏螺旋体基因型,构建系统发育树。结果显示：在147份鼠类样本中,经实时荧光定量PCR和巢式PCR方法检测,分别检出伯氏疏螺旋体阳性样本11份和13份,阳性率分别为7.48%和8.84%;13份巢式PCR阳性样本中,吉木萨尔县检出1份,新源县检出1份,乌苏市检出11份,通过序列同源性分析确定Borreliella garinii、Borreliella bavariensis为主要致病基因型。结果表明,北疆地区鼠类中存在伯氏疏螺旋体的自然感染,有传播给家畜及人群的风险,应加强该病原体检测和莱姆病防控。     

采用PCR方法对我国蜱伯氏疏螺旋体感染的流行病学调查

通过调查我国已有的4种蜱伯氏疏螺旋体的感染情况,以进一步阐明莱姆病在中国的分布与流行状况。本研究通过筛选扩增伯氏疏螺旋体外膜蛋白A(OspA)基因片段的通用引物,获得1对特异性引物,优化反应条件后建立用于检测蜱体内伯氏疏螺旋体的PCR方法,其扩增片段大小为307 bp。该方法可检测出10 pg的阿氏疏螺旋体(Ba)、1 pg的伽氏疏螺旋体(Bg)和0.01 pg的狭义伯氏疏螺旋体(Bb)的3种不同基因型的伯氏疏螺旋体的OspA基因组DNA,表明其敏感性较好,适用于蜱感染伯氏疏螺旋体状况的调查。本研究从我国7个省采集到的667只蜱,进行伯氏疏螺旋体感染的流行病学调查,分类鉴定表明这些蜱分属革蜱属、血蜱属、牛蜱属和扇头蜱属。PCR检测所获数据表明它们的感染率分别为4%(10/264)、6%(11/137)、31.4%(59/185)和31%     

含伯氏疏螺旋体OspA、OspC蛋白VLPs的构建及鉴定

莱姆病是一种经蜱传播的人兽共患病,随着人们经济的发展,伴侣动物的不断出现增加了人与动物经蜱传播莱姆病的风险,而当前并未有治疗莱姆病的有效方法,因此研制莱姆病疫苗成为一大热点。为了更好地预防莱姆病的发生,本研究通过替换新城疫病毒血凝素-神经氨酸酶蛋白(haemagglutinin-neuraminidase protein,HN)胞外域的方式,利用昆虫杆状病毒表达系统分别构建了含伯氏疏螺旋体候选抗原表位外膜蛋白A(outer surface protein A,OspA)、外膜蛋白C(outer surface protein C,OspC)的新城疫病毒样颗粒(virus-like particles,VLPs),形成了含有伯氏疏螺旋体抗原表位的新城疫病毒样颗粒,为之后研制莱姆病疫苗提供了新思路、新想法。     

伯氏疏螺旋体TaqMan-MGB探针荧光定量PCR方法的建立及对新疆地区蜱的检测

为建立一种TaqMan-MGB探针实时荧光定量PCR方法,来快速、特异地检测伯氏疏螺旋体,根据GenBank发表的伯氏疏螺旋体16SrRNA序列,应用分子生物学软件进行比对,选取保守序列设计特异性引物及TaqMan-MGB探针,并优化荧光定量PCR反应体系及条件,分别对大肠杆菌、沙门氏菌及钩端螺旋体进行扩增;构建伯氏疏螺旋体标准株16SrRNA基因片段阳性质粒,10倍系列稀释后进行real-time PCR扩增,测定该方法的敏感性。结果可见:仅伯氏疏螺旋体出现阳性扩增,而大肠杆菌、沙门氏菌及钩端螺旋体扩增均为阴性;该方法对阳性质粒的检测限约为100 copies/μL。应用该方法对新疆地区的100份蜱DNA样本进行检测,发现7份为伯氏疏螺旋体阳性。结果表明,本研究建立的TaqMan-MGB探针荧光定量PCR方法具有较高的特异性及良好的敏感性,为今后伯氏疏螺旋体的快速诊断及流行病学调查提供了技术支撑。     

牡丹江野生鹿血中伯氏疏螺旋体分子检测

为了解牡丹江地区野生鹿自然感染伯氏疏螺旋体的情况,2019年6月间在牡丹江海林市采集20份野生鹿血标本,应用普通PCR对鹿血标本进行5S-23S基因片段的扩增和测序,并将所得序列与GenBank中的项序列进行分析.结果检出7份阳性样本,阳性率为35％,序列分析发现伯氏疏螺旋体在有2个基因型,分别是Borreliella afzelii和Borreliella garinii.研究结果表明,该地区野生鹿存在伯氏疏螺旋体自然感染.     

伯氏疏螺旋体鞭毛基因的PCR单链构象多态性分析

应用多聚酶链反应单链构象多态性（ＰＣＲ－ＳＳＣＰ）技术分析了甘肃地区伯氏疏螺旋体和国际标准株Ｂ３１的鞭毛基因,发现甘肃不同宿主的２株伯氏疏螺旋体的鞭毛基因是一致的,属间一型螺旋体,而与Ｂ３１株明显不同,证明甘肃地区分离株与Ｂ３１株在鞭毛基因上不属同一型螺旋体。     

动物菜姆病的研究进展

莱姆病是由伯氏疏螺旋体引起的一种蜱传性人兽共患传染病。1977年美国医生A．C Sleere发现康涅狄格州莱姆镇流行的青少年关节炎是一种独立的疾病,并称为莱姆关节炎。1982年,昆虫学家Willy Burgdorfer从采自疫区的达敏硬蜱中发现和分离出莱姆病疏螺旋体。1984年Johnson根据其基因型和表型特征,认为该螺旋体是一个新种,命名为伯格多弗疏螺旋体。     

牛病毒性腹泻—黏膜病病毒C24V株E0截短基因密码子优化及其在卡介苗中的表达

The protein encoded by E₀ gene is one of the major protective antigens of bovine viral diarrhea-mucosal disease virus (BVDV). E₀ gene is a candidate for developing genetic engineering vaccine of BVDV and it could not express in BCG, it has been shown that E₀ protein has RNase activity, and has a plurality of rare codons according to analyze the codon usage frequency of BCG. By truncating the gene and optimizing its codons, E₀ gene expressed in BCG successfully and its antigenic activity was detected, which laid the foundation for building bivalent genetic engineering vaccine to prevent tuberculosis and bovine viral diarrhea-mucosal disease at the same time.     

新城疫病毒Anhinga株HN基因的克隆与生物信息学分析

克隆新城疫病毒中等毒力株Anhinga(Anh)的血凝素-神经氨酸酶(HN)基因,用生物信息学软件分析其编码蛋白结构与功能的差异.根据已知的HN基因序列,通过Primer Premier 5.0设计引物,提取新城疫病毒总RNA,反转录成cDNA,PCR扩增HN基因,并将其连接到T载体上进行测序比对;用DNA-man、D...     

PRRSV分离株GP3蛋白ORF3基因克隆及序列分析

为研究宁夏地区猪繁殖与呼吸综合征(PRRS)的流行规律,应用RT-PCR的方法从PRRSV分离株GP3蛋白中扩增出ORF3基因cDNA片段。结果显示,ORF3基因全长765 bp,编码254个氨基酸,与北美型毒株VR-2332的同源性达到85%以上,与欧洲型毒株LV的同源性低于60%,与国内近3年主要流行毒株JXA1、HEB1、HUB2亲缘性较近。对PRRSV分离株的亲水性分析表明,GP3蛋白的前57位氨基酸为疏水性信号肽序列;对PRRSV分离株GP3蛋白抗原表位分析表明,与国内外其他毒株抗原表位预测的结果基本一致,说明PRRSV分离株GP3蛋白具有与其他毒株相近的抗原特性。     

基于H蛋白基因的犬瘟热病毒遗传变异分析

犬瘟热是犬瘟热病毒(Canine distemper virus,CDV)感染犬和其他食肉动物造成的多发性、致死性传染病,本文从分子水平上探讨CDV遗传进化特性、变异情况与流行规律之间的关系.通过收集2002-2010年在中国地区分离的14株CDV野毒株、2006-2007年在全球各地分离的12株CDV野毒株以及从不同宿主分离的12株CDV野毒株和4株疫苗株,将其分为4组,将前3组野毒株分别与国内外正在使用的4株疫苗株的H基因进行遗传变异分析.分析发现,CDV野毒株与疫苗株间H蛋白基因的核苷酸相似性为86.2％～92.1％,其氨基酸相似性为89.1％～91.9％;H基因的584位的天冬酰胺糖基化位点是Asia-Ⅰ型CDV所特有的;H蛋白3555区域的非同义氨基酸替换概率较高.作者推测H蛋白抗原变异可能造成弱毒疫苗免疫效力降低,不能为某些CDV株的感染提供完全有效的保护.     

肺炎克雷伯菌榆中分离株OmpK17基因的克隆、生物信息学分析及免疫原性研究

试验旨在研究肺炎克雷伯菌(Klebsiella pneumoniae)外膜蛋白OmpK17基因序列特征及其免疫原性。参照GenBank中肺炎克雷伯菌外膜蛋白OmpK17基因序列(登录号:U52843.1)设计1对特异性引物,应用PCR技术扩增获得了肺炎克雷伯菌榆中分离株(YZ株)OmpK17基因CDS序列,应用相关软件及程序对OmpK17基因核苷酸序列及其推导的氨基酸序列进行生物信息学分析,构建其原核表达载体pET-OmpK17,并在大肠杆菌BL21(DE3)中进行了表达。表达产物纯化后免疫新西兰兔制备多抗血清,应用ELISA和Western blotting分析了重组蛋白的免疫原性。结果显示,肺炎克雷伯菌YZ株外膜蛋白OmpK17基因高度保守,其CDS序列全长513 bp,编码170个氨基酸,与GenBank中其他肺炎克雷伯菌OmpK17基因序列同源性均高于99.6%;OmpK17蛋白分子质量约为18.5 ku,具有较强的亲水性,含有1个信号肽、1个跨膜区和6个抗原决定簇区域,是一种具有桶状三维结构的跨膜蛋白。SDS-PAGE结果显示,OmpK17基因在大肠杆菌中获得表达且表达产物主要以包涵体形式存在。ELISA及Western blotting结果表明,肺炎克雷伯菌OmpK17蛋白具有良好的免疫原性。本研究克隆和表达了肺炎克雷伯菌OmpK17基因及其编码蛋白,并证实该蛋白具有良好的免疫原性,为OmpK17基因的生物学特性研究和肺炎克雷伯菌检测方法的建立奠定了基础,为肺炎克雷伯菌基因工程疫苗的研发提供了理论依据。     

牛支原体新疆分离株P48基因克隆及分子特征分析

本研究旨在分析牛支原体P48基因的序列特性、蛋白的结构和功能。根据GenBank中PG45菌株P48基因序列（GenBank登录号：CP002188.1）设计特异性引物,运用Overlap-PCR扩增获得P48基因全长,其目的片段连接到pMD19-T载体上并进行测序,利用生物信息学方法分析和预测其核苷酸序列及其编码蛋白的理化性质、结构功能（信号肽、跨膜结构、磷酸化位点、糖基化位点、二级结构和三级结构）。结果显示,分离株P48基因序列为1 407 bp,与Mb PG45国际标准株的同源性为100%,聚类于一支;P48基因编码467个氨基酸残基,组成一个无跨膜、稳定的亲水性蛋白;P48蛋白存在信号肽,有44个潜在的磷酸化位点和1个糖基化位点,其二级结构是混合型,其中α-螺旋所占比例最高（41.76%）,无规则卷曲次之（37.69%）。功能预测显示,在信号转导、胁迫应答、受体等方面该蛋白存在较高概率。本研究成功克隆了分离株P48基因片段,其编码的蛋白是一个稳定可溶、亲水性蛋白,为分析分离株P48基因的特性和功能提供了一定的理论基础和科学依据。     

Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene

The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.     

Nucleotide sequence of a gene encoding a new genus specific protein of Chlamydia psittaci.

DNA fragment No. 13 from the C. psittaci pigeon strain, has been cloned in the plasmid pUC19. Hybridization analysis revealed that the fragment maintained a chlamydial common sequence. Furthermore, nucleotide sequencing identified two partial open reading frames (ORF), 675b. p. and 530b. p. Expression of ORFs revealed that the second ORF encoded 25KD polypeptide, whereas the first ORF did not produce any antigenic product. The 25KD beta-galactosidase fusion protein reacted strongly with chlamydia-specific antibodies elicited against a number of different chlamydial strains. Gene Bank analysis showed that this cloned gene is not highly homologous with chlamydia or other organisms for which nucleotide sequences have already been published. This 25KD polypeptide may be an additional genus-specific antigen of Chlamydiae.     

Investigation of SnSPR1, a novel and abundant surface protein of Sarcocystis neurona merozoites

An expressed sequence tag (EST) sequencing project has produced over 15,000 partial cDNA sequences from the equine pathogen Sarcocystis neurona. While many of the sequences are clear homologues of previously characterized genes, a significant number of the S. neurona ESTs do not exhibit similarity to anything in the extensive sequence databases that have been generated. In an effort to characterize parasite proteins that are novel to S. neurona, a seemingly unique gene was selected for further investigation based on its abundant representation in the collection of ESTs and the predicted presence of a signal peptide and glycolipid anchor addition on the encoded protein. The gene was expressed in E. coli, and monospecific polyclonal antiserum against the recombinant protein was produced by immunization of a rabbit. Characterization of the native protein in S. neurona merozoites and schizonts revealed that it is a low molecular weight surface protein that is expressed throughout intracellular development of the parasite. The protein was designated Surface Protein 1 (SPR1) to reflect its display on the outer surface of merozoites and to distinguish it from the ubiquitous SAG/SRS surface antigens of the heteroxenous Coccidia. Interestingly, infection assays in the presence of the polyclonal antiserum suggested that SnSPR1 plays some role in attachment and/or invasion of host cells by S. neurona merozoites. The work described herein represents a general template for selecting and characterizing the various unidentified gene sequences that are plentiful in the EST databases for S. neurona and other apicomplexans. Furthermore, this study illustrates the value of investigating these novel sequences since it can offer new candidates for diagnostic or vaccine development while also providing greater insight into the biology of these parasites.     

Characterization of flagellin from Clostridium chauvoei.

Differential centrifugation and cesium chloride-equilibrium centrifugation were used to purify the flagella from the strain Okinawa of the formalin-fixed Clostridium chauvoei. SDS-PAGE profile of purified flagella showed that a major protein band with a molecular mass of 46 kDa, corresponding to the flagellin monomer, and at least two minor protein bands with molecular masses of approximately 73 and 100 kDa were found. The amino acid composition of C. chauvoei flagellin was similar to the flagellin of Salmonella typhimurium and Bacillus subtilis. In addition, C. chauvoei flagellin monomer shared limited sequence homology with the N-terminal amino acid sequence reported for other bacterial flagellins. N-terminal sequences of two minor bands corresponded to the flagellin monomer, indicating that higher molecular mass bands were polymeric forms of the flagellin monomer.