小肠结肠耶氏菌毒性质粒基因文库的构建

本研究以pAT153质粒为载体,构建了小肠结肠耶氏菌毒性质粒(pVYE)经限制性内切酶Bam HI和PstI消化产生的DNA片段的基因文库.实验克隆了8株(pYBI～8)pVYE的BamHI片段和6株(pYPI～6)pVYE的PstI片段的重组子.其中pYB4、pYB7、pYB8重组质粒中插入的pVYE—Bam HIDNA片段的分子量分别为8.7、3.8、20kb;pYP3、pYP5、pYP6重组质粒中插入的pVYE-Pst I DNA片段的分子量分别为4.5、3.0、7.0kb.本实验结果为进一步筛选和制备特异性基因探针奠定了基础.     

《兽医大学学报》1992年第12卷总目次

第1期禽多杀性巴氏杆菌保护性荚膜抗原基因的克隆及表达………………………………………姚湘燕等(l)大肠杆菌K。。。与LT(A-B”)抗原基因质粒在猪霍乱沙门氏菌弱毒株中的表达—………·。··韩文瑜等(7)强毒小肠结肠耶氏菌生物素标记基因探针的研究—…………………………………………·黎诚槽等(互二)制备性分段电泳纯化基因工程白细胞介素一2……………··,…,…………………………··赵建中等(18)草鱼～鲤的细胞核移植—…………………………………·。………。…………·,……·,………齐福印等(22)光照对鸡肝、胰酶组织化学的影响…     

生物素标记LT基因探针检测产肠毒素性大肠杆菌

用碱变性法提取重组质粒pEWD299,经Hind Ⅲ酶切后,进行琼脂糖凝胶电泳,用电洗脱法回收LT基因850bp的核酸片段,再用二步法缺口翻译反应制备生物素标记的LT基因探针.通过菌落杂交试验表明,在严格去蛋白和RNA的条件下,该探针与试验中所有产LT和ST,或仅产LT的ETEC发生杂交反应;而与所有仅产ST的ETEC、侵袭性大肠杆菌(EIEC)、普通大肠杆菌、沙门氏菌、志贺氏菌、小肠结肠耶尔森氏菌、霍乱弧菌均无杂交反应.其检测提纯的LT—DNA的敏感性水平为25pg,检测食品模拟标本中产LT大肠杆菌的敏感性达到130个细菌/g的水平.     

在普通培养基上区别小肠结肠耶氏菌毒力株与无毒株

小肠结肠耶氏菌分布广泛,已从多种动物及食品中分离到该菌。由该菌毒力株引起的腹泻也屡见暴发流行的报道。因此区别小肠结肠耶氏菌毒力株及无毒株极其重要。但目前报道的一些区别方法都存在一些问题。为此,我们参照Saumya Bhaduri等报道的区别毒力株和无毒株的结晶紫结合试验,将脑心浸液培养基改为普通培养基,经初步试验取得较好效果。现报     

鸡传染性喉气管炎病毒核酸探针的制备及特异性鉴定

从鸡传染性喉气管炎病毒王岗株感染的鸡胚绒毛尿囊膜细胞中提取病毒,并抽提其核酸,经限制性核酸内切酶Pst Ⅰ完全消化后,在0.7%琼脂糖凝胶中电泳分离,然后用DE—81滤纸回收2～6kb区域的片段与质粒pBluescrip~+SK重组.有3个重组质粒(pLT—1,pLT—2和pLT—4)所含外源DNA片段的大小,分别与4.3kb,4.8kb和1.6kb的鸡传染性喉气管炎病毒DNA Pst Ⅰ片段一致,经过Southern杂交试验证明,这3个片段都是病毒DNA的特异性片段.用光生物素标记重组质粒pLT—1和pLT—2后混合作为探针,再与鸡喉气管炎病毒及其他8种鸡传染性病原进行杂交,证明此探针只与喉气管炎病毒反应,而与其他8种病原无任何交叉反应.     

新城疫病毒融合蛋白裂解位点基因光敏生物素探针的制备及初步应用

将重组质粒ｐＵＣＬａＦｃ和ｐＵＣＦ４６Ｆｃ用ＥｃｏＲＩ和ＳａｌⅠ酶切,回收新城疫病毒融合蛋白裂解位点（Ｆｃ）基因,将此Ｆｃ片段用光敏生物素标记,制备出Ｆｃ探针。该探针能够与新城疫病毒（ＮＤＶ）的强毒株Ｆ４６Ｅ９、中毒株Ｍ株和弱毒株ＬａＳｏｔａＥ４株杂交,而不与对照的传染性支气管炎病毒（ＩＢＶ）、减蛋综合征（ＥＤＳ－７６）病毒杂交,说明探针是特异的。用ｐＵＣＬａＦｃ的Ｆｃ探针检测了各５份ＮＤＶ强中弱毒株的尿囊液,均为阳性。但强毒株和弱毒株的Ｆｃ探针都能与所用的强、中、弱毒株杂交,说明该Ｆｃ探针尚不能区分强、弱毒株。     

鸡痘病毒282E_4弱毒株TK基因的定位与克隆

将鸡痘病毒282E_4弱毒疫苗株的基因组分别进行Eco RI、Hind III和Bam HI酶切,以地高辛-dUTP标记澳大利亚鸡痘病毒疫苗株的TK基因作探针,进行Southern印迹杂交.结果发现,鸡瘟病毒282E_4弱毒株TK基因位于5.5kb Eco RI、3.7kb Hind III和4.3kb Bam HI酶切片段中.随后回收3.7kb Hind Ⅲ片段,克隆到pUC18质粒中.经Southern印迹杂交证实,已成功地克隆了我国鸡痘病毒282E_4弱毒株TK基因.     

马立克氏病病毒光敏生物素核酸探针的制备及应用

从马立克氏病病毒(MDV)GA株Bam HI基因文库中选取L片段,用光敏生物素标记该片段,制备了MDV光敏生物素核酸探针。其检测灵敏度达Pg水平,保存期至少4个月。对细胞培养物中病毒核酸的检测结果表明,探针只与血清Ⅰ型MDV DNA发生分子杂交,而不与血清Ⅱ型(SB-1)和血清Ⅲ型(HVT)MDV DNA发生反应。分别以京-1株(血清Ⅰ型)和Fc126株(血清Ⅲ型)接种1日龄雏鸡,于14日龄和30日龄采用斑点杂交法检测羽髓提取的病毒DNA,结果京-1株均为阳性,Fc126株均为阴性,表明该探针可以用于MDV强毒株和HVT疫苗毒株的区别诊断。采用斑点杂交法检测实验感染MDV GA株的鸡全部为阳性(16/16),检出率为100％;而用琼脂免疫扩散试验的检出率为87.5％(14/10)。试验表明,所制备的MDV光敏生物素核酸探针具有特异性强、灵敏度高、无放射性污染等优点。为MDV强毒株的临床检测及MDV分子生物学研究提供了一种重要手段。     

马立克氏病病毒光敏生物素核酸探针的制备及应用

从马立克氏病病毒（MDV）GA株BamHI基因文库中选取L片段，用光敏生物素标记该片段，制备了MDV光敏生物素核酸探针，其检测灵敏度pg水平，保存期至少4个月，对细胞培养物中病毒核酸的检测结果表明，探针只与血清Ⅰ型MDV DNA发生分子杂交，而不与血清Ⅱ型（SB－1和血清Ⅲ型（HVT（MDV DNA发生反应，分别以京－1株（血清Ⅰ型）和Fc126株（血清Ⅲ型）按种1日龄急诊鸡，于14日龄和30日龄采用斑点杂交法检测须提取的病毒DNA，结果京－1株均为阳性，Fc126株均为阴性，表明该探针可以用于MDV强毒株和HVT疫苗毒株的区别诊断，采用斑点发校法检测实验感染MDV GA株的鸡全部为阳性（16／16），检出率为100％，而用琼脂免疫扩散试验的检出率为87．5％（14／16）。结果表明，所制备的MDV光敏生物素核酸探针具有特异性强、灵敏度高、无放射性污染等优点，为MDV强毒株的临床检测及MDV分子生物学研究提供了一种重要手段。     

用马立克氏病病毒强毒的BamH-L片段核酸鉴定血清1型毒株

马立克氏病病毒 (MDV) GA强毒株的 Bam H - L 片段 DNA全长为 2 892 bp,G C比率为 5 5 .91%。用内切酶将其亚克隆为 6个片段 :Bam H - Pst 、Pst - Pst 、Pst - Cla 、Cla - Sm a 、Sm a - Sma 和 Sm a - Bam H 。用地高辛标记 L 片段 DNA或者分别标记其 6个亚片段核酸 ,通过斑点杂交试验都可将 MDV血清 1型毒株与 2型和 3型的毒株区分开来 ,应用 PCR方法可获得相同结果。但 PCR方法更敏感、更快捷。结果表明 ,MDV GA强毒株的Bam H - L片段 DNA对 MDV血清 1型毒株是特异的     

The relationship of plasmids from environmental Yersinia isolates and the virulence plasmid of enteropathogenic Yersinia strains

The human pathogenic strains of Yersinia harbour a conserved plasmid carrying the Yop virulon. The virulence plasmid of Yersinia enterocolitica strains belonging to the serogroups O:3 and O:9 were used as probes to detect homologous sequences in plasmids of "avirulent" Yersinia strains. "Avirulent" Yersinia strains (Y. enterocolitica biogroup 1A, Y. intermedia, Y. kristensenii and Y. frederiksenii) lack the virulence plasmid. They are widely distributed in the environment and can frequently be isolated from clinical samples. Hybridisation experiments revealed a number of common genetic elements of the virulence plasmid and the plasmids of "avirulent" Yersinia strains. These elements were identified as genes involved in plasmid replication, as an endonuclease gene and as mobile genetic elements. However, none of the plasmid encoded virulence genes was present in the plasmids of "avirulent" Yersinia strains. The frequent occurrence and the possible etiological relevance of "avirulent" isolates will be discussed.     

Differentiation between virulent and avirulent strains of infectious laryngotracheitis virus by DNA:DNA hybridization using a cloned DNA marker

The DNAs of virulent and avirulent strains of infectious laryngotracheitis virus (ILTV) showed greater than 96% homology by reciprocal DNA:DNA hybridization. Nevertheless, by use of selected restricted DNA fragments, it was possible to differentiate strains according to pathotype more readily than by the more laborious restriction enzyme analysis. Restricted DNA fragments were successfully cloned into Escherichia coli HB101 cells and could be used not only for pathotyping ILTV strains but also for their differentiation from other avian viruses.     

多重聚合酶链反应快速检测鉴别鸡毒支原体强毒株和弱毒疫苗株的研究

根据鸡毒支原体强毒株和弱毒疫苗株基因组的结构特点，设计合成了二对引物XZ1，XZ2和XZ45、XZ46，建立了一种同时检测鉴别MG野毒株和弱毒疫苗株的多重PCR技术。试验结果表明，用这两对引物对MG强毒株和弱毒疫苗侏进行多重PCR，强毒株只扩增出732bp一条带，而弱毒疫苗株则可同时扩增出732bp、524bp二条带，而对其他种类鸡支原体和其它禽病病原的扩增不出现任何条带，结果均为阴性；敏感性测定结果表明，该多重PCR最低能检出1Pg的MG强毒株和弱毒疫苗株的DNA模板。     

Identification and distribution of putative virulent genes in strains of Streptococcus suis serotype 2

In order to identify gene sequences unique to the virulent strains, suppression subtractive hybridization (SSH) was conducted using virulent Streptococcus suis type 2 (SS2) strain HA9801 and avirulent S. suis type 2 strain T15. Thirty genomic regions were absent in T15, and the DNA sequences of these regions in HA9801 were determined. These DNA fragments, containing putative virulence genes, encoded 28 proteins that were homologous to proteins involved in various aspects of cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems and others of unknown function. According to the published SS2 genomic sequence of the Chinese strain 98HAH33, PCR primers for 14 significant DNA fragments were designed and used for detection of the distribution of these fragments in S. suis strains from different sources, serotypes, regions, groups and times. The results showed that these 14 DNA fragments were widely distributed in 37 detected SS2 strains, yet were absent among the avirulent strain T15. Moreover, these fragments could be detected in other serotypes of S. suis, but each serotype had a different distribution of the fragments.     

The application of PCR to the identification of selected virulence markers of Yersinia genus

The subject of this study was thirty nine strains of Yersinia enterocolitica, isolated from faeces of humans who showed symptoms typical of intestinal yersiniosis, and seventy strains of Y enterocolitica, four strains of Y. pseudotuberculosis, and one strain of Y. kristensenii from healthy pigs. In the population tested the following serogroups appeared: O3, O9, O2, O5. A PCR was used to detect the presence of pathogenic chromosomal markers, such as myfA and inv genes of the tested Yersinia species. Among Y. enterocolitica strains isolated from humans and belonging to serogroup O3 (thirty four strains) and serogroup O9 (five strains) thirty three Y. enterocolitica O3 strains and four Y. enterocolifica O9 strains, gave a positive reaction to the nmyfA gene, yielding a fragment of 280 base pairs (bp). Among seventy Y. enterocolitica strains isolated from pigs forty strains belonging to serogroup O3 and fifteen strains belonging to serogroup O9 gave a positive reaction to the myfA gene. The presence of 390 bp amplified products, corresponding to the inv gene fragment, was detected in PCR products of three Y pseudotlluberculosis strains from pigs and only in one Y. enterocolitica O3 strain from humans, which had no myfA gene. The results obtained show that the myfA gene is only present in the strains that belong to pathogenic serotypes of Y. enterocolitica. The myfA gene prevailed in the Y. enterocolitica O3 and O9 strains from humans but was less common in the Y. enterocolitica O3 and O9 strains from pigs.     

Recombinant DNA probes for Mycoplasma synoviae

A genomic library was prepared from Mycoplasma synoviae (MS) strain WVU 1853 cloned in plasmid vector pUC8 and transformed in Escherichia coli host JM83. In dot blot assays, four transformed E. coli clones hybridized with 32P-labeled chromosomal DNA of MS but not with 32P-labeled chromosomal DNA of M. gallisepticum (MG) strain S6. In Southern hybridization, each of the CsCl-purified recombinant plasmid clones was shown to contain two MS DNA fragments between 1.0 to 2.3 kbp in length. 32P-Labeled probes prepared from each of the four recombinant plasmids hybridized in dot blot assays with MS strain WVU 1853 and nine MS field isolates but not with MG strains S6, K810, F2F10, four MG field isolates, and 15 other species of avian mycoplasmas.     

新城疫病毒F48E8株融合蛋白基因的克隆

以提纯的我国标准ＵＤＶＦ４８Ｅ８强毒株基因组ＲＮＡ为模板、化学合成的融合蛋白（Ｆ）基因特异寡核苷酸为引物，反转录合成Ｆ基因ｃＤＮＡ，并采用同聚物加尾的方法克隆到细菌质粒ｐＧＥＭ３Ｚｆ（－）。经ＡＩＸ平板筛选和酶切分析，并用Ｆ基因片段核酸探针作ｄｏｔ－ｂｌｏｔ检测，共获得插入片段长度在０．６￣２．８ｋｂ之间的阳性克隆３４个，其中插入片段大于１．５ｋｂ的阳性克隆８个。用多种限制性内切酶对阳性克隆ｐＦ７     

Development of a recombinant Anaplasma marginale DNA probe

An Anaplasma marginale DNA probe has been developed by using an improved method for the isolation of genomic DNA. Purified genomic A. marginale DNA from the St. Croix isolate was partially digested with Sau 3A1 into fragments (greater than or equal to 5.0 kb). The restriction fragments were cloned using standard techniques in the pBR322 vector and used to transform E. coli (DH5) host cells. The recombinant A. marginale DNA library was screened by the colony lifting procedure. Colonies containing plasmids with A. marginale DNA inserts were identified by hybridization with a genomic A. marginale DNA radiolabeled probe (32P). Seven recombinant A. marginale DNA probes were evaluated by dot-blot in vitro hybridization assays to identify candidates as diagnostic tools in bovine anaplasmosis studies. Specificity and sensitivity experiments were carried out by using heterologous and homologous DNAs. The heterologous panel contained bovine DNA (WBC) and blood parasites DNA from Babesia bovis (Bb), Babesia bigemina (Bbi), Eperythrozoon suis (Es) and Eperythrozoon wenyoni (Ew). The homologous DNA panel included A. marginale DNAs of 12 different isolates which were isolated in the Caribbean, Mexico, and the U.S.A. The selected diagnostic probe was identified as pSt. Croix A1, and labeled with 32P by using in vitro nick translation and random primer techniques. The pSt. Croix A1 probe demonstrated 100% specificity and high sensitivity by hybridization in dot blotting and Southern blotting. The probe can detect 500-1000 infected erythrocytes per microliters which corresponds to a parasitemia of less than 0.01%. The A. marginale DNA insert was approximately 6.4 kb in size and a partial restriction map has been constructed.     

血清Ⅱ型鸭疫里默氏杆菌基因组文库的构建

为研究血清Ⅱ型鸭疫里默氏杆菌的抗原基因,筛选毒力较强的血清Ⅱ型鸭疫里默氏杆菌菌株,提取其基因组,分别用Sau3A I酶切法和shotgun法对基因组进行处理后,选取1.5～3 kb的DNA片段,与PUC118载体连接后电转化JM109大肠杆菌感受态细胞,构建血清Ⅱ型鸭疫里默氏杆菌基因组文库。两种方法得到的库容量分别为40 000和50 000左右,随机挑选阳性克隆鉴定表明有外源DNA片段的插入,说明建库成功。该文库的成功构建为Ⅱ型鸭疫里默氏杆菌新的抗原基因的筛选奠定了基础。