强毒小肠结肠耶氏菌生物素标记基因探针的研究

在构建的小肠结肠耶氏菌毒性质粒DNA基因文库pYB1～8与pYP1～6的基础上,筛选出了pYB7和pYP6克隆株.用限制性内切酶Bam HI消化pYB7,Pst消化pYP6,可分离出3.8kb和6.4kb的插入性DNA片段.以这两个基因片段为目的基因,用生物素化dUTP和光敏生物素标记,获得了生物素标记的基因探针.该探针能检出10pg以上的强毒小肠结肠耶氏菌DNA,不与无毒小肠结肠耶氏菌及大肠杆菌、鼠伤寒沙门氏菌、金黄色葡萄球菌等18种对照菌反应,具有高度的特异性和敏感性.pYB7与pYP6探针对不同血清型及来源的小肠结肠耶氏菌检测,其结果与自凝性试验、依钙试验等结果相符;对小肠结肠耶氏菌强毒株与无毒株检定的准确率为100%.     

禽多杀性巴氏杆菌保护性荚膜抗原基因的克隆及表达

提取禽源多杀性巴氏杆菌C48—1的染色体,用“鸟枪法”将酶解染色体DNA片段重组到pBR322质粒载体的Pst I酶切点上,再转化到大肠杆菌RRI中.经耐药表型筛选,在约5000个转化菌中,找出了887个具有重组质粒的细菌株,其一般细菌学性质与大肠杆菌RRI相同,构成了禽多杀性巴氏杆菌C48—1染色体DNA的不完全基因文库.分离提纯保护性荚膜抗原,制备抗血清,用~(125)I SPA固相放射免疫技术,从文库中选出13株阳性表达菌株,与用ELISA法复试结果吻合,其中10号和696号菌株表达较强.对10号和696号菌进行了动物试验,结果1次免疫小鼠分别获3/7、2/7的保护,2次免疫分别获6/11和5/11的保护,与之对照的阴性转化菌(119号)以及大肠杆菌(RRI)则无保护力(0/7、0/10).用快速细菌破碎法对13个阳性菌株进行了检测,其外源基因片段在0.5～5.4kb之间.对10号阳性菌免疫电镜观察,在细胞膜及细胞浆内均可见到高电子密度区,显示出外源基因表达的位置和程度.10号菌的重组质粒命名为PPM10,分子量约为5.16kb,用Pst I酶切PPM10,得到约4.3kb和0.86kb的两个片段,因此插入片段约0.86kb.进一步用内切酶分析,证明该插入片段上无Bam HI、Eco RI、Hind III和另外的Pst I酶切点.     

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.     

鸡致病性大肠杆菌O1、O2和O78的Ⅰ型菌毛结构基因的克隆、序列测定及同源性分析

提取鸡致病性大肠杆菌分离株O1、O2和O78的基因组DNA作为模板,用TD-PCR技术从其中分别扩增出了0.55kb的Ⅰ型菌毛结构基因(piliA).将扩增得到的piliA基因片段,用TA克隆的方法分别克隆进pGEM-T载体中,转化至受体菌JM109中,用Amp/IPTG/X-gal琼脂平板蓝白菌落筛选法,得到含阳性重组子的菌株,提取质粒用PstI单酶切及NcoI和PstI双酶切鉴定,结果证实,所构建的克隆质粒中均含有相应piliA基因.经DNA序列分析,其结构基因阅读框架大小为549 bp,但其中O1菌株Ⅰ型菌毛基因在第72位发生突变,有6个碱基插入.经DNA Star核酸分析软件分析,此3菌株的Ⅰ型菌毛基因的同源性为89.9%～92%.     

伪狂犬病病毒Ma株基因组3.4kb及4.4kbBamHI片段的克隆与鉴定

本试验采用BamHI酶切伪狂犬病病毒 (PRV)Ma株基因组DNA ,电泳回收 3 4kb和4 4kb片段 ,快速克隆了伪狂犬病病毒Ma株BamHI 3 4kb和 4 4kb片段到质粒 pUC1 9中 ,对其进行部分序列测定并与Genebank序列进行同源性分析 ,结果表明BamHI 3 4kb片段包含UL50等基因 ,与Ka株UL50基因同源性为 87% ,BamHI 4 4kb片段包含RSP40及PK等基因片段 ,与Ka株RSP40基因同源性为 98%。Ka株BamHI 3 4kb片段包含TK基因 ,而包含UL50基因的BamHI片段为 7 4kb。结果表明 ,PRVMa株BamHI酶切位点发生了漂移     

瘤胃中小韦荣球菌H2基因文库的构建

为了构建小韦荣球菌H2基因的文库,研究提取小韦荣球菌H2总基因组,采用机械剪切方法获得DNA随机片段,将获得的DNA片段与Sma Ⅰ酶切质粒pUC18体外连接并电转化至大肠杆菌DH5α,在含有IPTG、X-gal和氨苄西林的LB琼脂平板上用蓝白斑法筛选含重组质粒的大肠杆菌,并进行扩增分析.结果表明:重组质粒经鉴定分析证实包含小韦荣球菌H2的3～4 kb插入片段,文库滴度为1×105 pfu/mL;根据公式N=ln(1-p)/(1-f)证明99%基因组包含在文库中.     

鸡致病性大肠杆菌O_1、O_2和O_(78)的Ι型菌毛结构基因的克隆、序列测定及同源性分析

提取鸡致病性大肠杆菌分离株O1、O2和O78的基因组DNA作为模板,用TD-PCR技术从其中分别扩增出了0.55kb的I型菌毛结构基因(piliA)。将扩增得到的piliA基因片段,用TA克隆的方法分别克隆进pGEM-T载体中,转化至受体菌JM109中,用Amp/IPTG/X-gal琼脂平板蓝白菌落筛选法,得到含阳性重组子的菌株,提取质粒用PstI单酶切及NcoI和PstI双酶切鉴定,结果证实,所构建的克隆质粒中均含有相应piliA基因。经DNA序列分析,其结构基因阅读框架大小为549bp,但其中O1菌株I型菌毛基因在第72位发生突变,有6个碱基插入。经DNAStar核酸分析软件分析,此3菌株的I型菌毛基因的同源性为89.9%~92%。     

伪狂犬病病毒PK基因的克隆与PK、gG双缺失转移载体的构建

地高辛标记伪狂犬病病毒（PRV）Ea株短区段蛋白激酶（PK）基因3′端0．4kb片段，Southern杂交确定短区段PK基因定位在基因组DNA BamH Ⅰ 4．0kb片段中。将该片段克隆获得重组质粒pSB304，对pSB304亚克隆，构建了仅含完整PK基因约1．3kb片段的重组质粒pSB305，并进行了序列测定。结果表明，PK基因存在2种可能的同框编码方式，分别编码388或334个氨基酸残基，并具有真核细胞蛋白激酶催化结构域序列。同国外PRV NIA-3、Ka株相比，氨基酸同源性分别为98．8％和97．3％，有意义的是Ea株、Ka株均较NIA-3株在同一位置缺失2个氨基酸（Asp，Gly）。进一步对pSB305和含gG全基因以及部分gD基因的质粒pUSK进行酶切拼接，将PK基因大部分编码区、gG基因5′端部分编码区进行缺失，构建成两端同源侧翼分别为3．1kb和1．6kb的PK、gG双缺失转移载体pLR001。上述结果为深入研究PK基因功能及研制更安全的TK^-／PK^-／gG^-三缺失基因工程疫苗奠定了基础。     

伪狂犬病病毒糖蛋白gp50基因的克隆和表达

从包含伪狂犬病病毒（ＰＲＶ）闽Ａ株ＢａｍＨＩ－７片段的重组质粒ｐＰＲ１２８中分离出含有完整糖蛋白ｇｐ５０基因的２．１ｋｂＤＮＡ片段，用ＫｐｎＩ和ＳｔｕＩ酶切后，将其酶切片段分别克隆到ｐＵＣ１９载体中，构建了２．１ｋｂ片段完整测序用质粒。对其序列进行分析，发现与文献报道结果一致，证明分离的ｇｐ５０基因是正确的。将包含ｇｐ５０基因的２．１ｋｂ和１．６ｋｂＤＮＡ片段分别插入带有痘苗病毒天坛株ＴＫ基因区段的ｐＧＪＰ－５质粒Ｐ７．５启动子的下游，构建了ｐＧＢＴ５０－３６和ｐＧＢＴ５０－Ｓ２２个嵌合载体。将嵌合载体通过磷酸钙共沉淀法转染预先感染ＴＫ＋痘苗病毒天坛株的人ＴＫ－１４３细胞或ＣＶ－１细胞，进行体内同源重组。经蚀斑纯化，在ＢｄＵＲ选择压力下，通过光敏生物素标记的探针杂交，获得带有ＰＲＶｇｐ５０基因的重组痘苗病毒。用ＥＬＩＳＡ检测，重组痘苗病毒有特异性ＰＲＶｇｐ５０抗原存在。     

伪狂犬病病毒通用转移载体的构建及应用

将来自质粒pFSV40的300bpBamHI/PstI片段[其中含有SV40poly(A)和部分多克隆位点]插入到质粒pUSK相应的酶切位点中，获得重组质粒USKSV40。该重组质粒中gG基因5’端编码区缺失了428bp。将来自质粒pcDNA3.1( )的946bpBglⅡEcoRI片段（其中含CMV启动子及部分多克隆位点）插入到质粒pUSKSV40的BamHIEcoRI位点，构建了通用载体pPRVCMV-uni，其中含有CMV启动子，SV40poly(A)以及NheI,Pme1,BamHI,BstXI,EcoRI,StuI,XbaI等7个单一克隆位点，将eGFP基因插入到该通用载体的BamHI和EcoRI之间，用所获得的转移载体与TK/gG-/LacZ^ PRV基因组共转染PK-15细胞，经检测eGFP基因在重组伪狂犬病病毒中获得表达，从而证实该通用载体的构建是可行的。本研究研制以伪狂犬病病毒为载体的二价或多价基因工程疫苗奠定了物质基础。     

PRV闽A株Bam HI片段克隆及其第7片段的鉴定

用鸟枪法将ＰＲＶ闽Ａ株的ＢａｍＨＩ酶切片断克隆到ｐＢＲ３２２质粒中，再经抗性筛选、酶切鉴定和菌落原位杂交，证实已克隆了ＰＲＶ闽Ａ株１４个ＢａｍＨＩ酶切片段中的１２个，从而构建了其基因文库，通过Ｓｏｕｔｈｅｒｎ转印杂交和酶切图谱分析鉴定了重组质粒ｐＰＲ１２８，其插入片段含量包含了ＰＲＶ闽Ａ株糖蛋白ｇｐ５０基因在内的ＢａｍＨＩ－７片段，核酸长约６．８ｋｂ。     

鸡痘病毒282E4弱毒株TK基因限制性酶切图谱分析

以限制性内切酶ＢａｍＨＩ、Ｘｂａｌ、Ｃｌａｌ、Ｈ１ｎｄⅢ、Ｎｃａｌ对含有鸡痘病毒（ＦＰＶ）２８２Ｅ４弱毒株３．７ｋｂＨｉｎｄⅢＴＫ基因片段的重组质粒ｐＳＬ１进行单酶和它们之间双酶酶切。结果表明：３．７ｋｂＨｉｎｄⅢＴＫ基因片段上有２个Ｃｌａｌ切点，２个Ｘｂａｌ切点，１个Ｎｃａｌ切点，没有ＢａｍＨＩ切点。随后，用澳大利亚ＦＰＶ２．２ｋｂＨｉｎｄⅢ＋ＣｌａｌＴＫ基因作探针，对各种酶切片段进行Ｓｏｕｔｈｅｒｎ印迹杂交，进一步确定ＴＫ基因位于２．２ｋｂＨｉｎｄⅢ＋Ｃｌａｌ片段中。杂交结果与限制性酶切图谱分析结果相一致。该基因的限制性酶切图谱与澳大利亚ＦＰＶ疫苗株的基本相同。     

DNA fingerprinting of Australian isolates of Mycobacterium avium subsp paratuberculosis using IS900 RFLP

OBJECTIVES: To evaluate additional restriction enzymes for IS900 RFLP of Mycobacterium avium subsp paratuberculosis and examine the genetic diversity among Australian isolates for epidemiological studies of Johne's disease. DESIGN AND PROCEDURE: Seventy-one isolates of M paratuberculosis from cattle, sheep, goat, alpaca and rhinoceros in six Australian States and the Northern Territory, reference strains and reference DNA from previously characterised strains were tested for genetic variation. Bst EII, Pvu II and Pst I restriction enzymes were used, and four others (Bam HI, Alu I, Xho I and Dra I) were assessed for their ability to detect polymorphisms. Multiple isolates from some animals were tested. RESULTS: Bam HI, was the most effective enzyme for identifying polymorphisms (12 types), followed by Bst EII (11 types). Both Pvu II and Pst I were relatively ineffectual. Fifteen different types were identified, 12 in clinical isolates. Most isolates were cattle (C) strains and fell into the C1 (n = 28) and C3 (n = 32) groupings. All isolates from alpaca were type C1, and bovine isolates were commonly C1 (n = 15) or C3 (n = 28). All of the sheep were infected with sheep (S) strains; no S strains were identified in cattle. Two of six isolates from one animal had single band differences. CONCLUSION: The epidemiological features of M paratuberculosis in Australia are similar to those reported in New Zealand, where cattle and sheep are commonly infected with different strains. However, because of the lack of polymorphism identified within the major groups, it is unlikely that DNA fingerprinting will have a significant role in epidemiological studies of Johne's disease, unless an unusual strain in being studied.     

伪狂犬病病毒鄂A株TK基因的克隆及其鉴定

合成了 １ 对能对伪狂犬病病毒（ Ｐｓｅｕｄｏｒａｂｉｅｓ ｖｉｒｕｓ, Ｐ Ｒ Ｖ） Ｔ Ｋ（ｔｈｙｍ ｉｄｉｎｅ ｋｉｎａｓｅ）基因＋ １１９～＋ １ ０７１区进行特异扩增的引物,用猪 Ｐ Ｒ Ｖ 鄂 Ａ 株细胞培养物提取的基因组作模板,扩增出 ９５３ ｂｐ 长的片段,用地高辛标记该片段作探针,通过 Ｓｏｕｔｈ ｅｒｎ 杂交,从克隆有 Ｐ Ｒ Ｖ 鄂 Ａ 株的 Ｂａｍ Ｈ Ｉ片段的重组质粒中钓出含 Ｔ Ｋ 基因的重组质粒 ｐ Ｓ Ｔ Ｋ。对 ｐ Ｓ Ｔ Ｋ 进行酶切分析,绘制了含 Ｔ Ｋ 基因的 Ｂａｍ Ｈ Ｉ片段的图谱,通过测序得出了 Ｔ Ｋ 基因的全序列。将该序列与 Ｐ Ｒ Ｖ Ｎ Ｉ Ａ３ 株 Ｔ Ｋ 基因进行比较,发现鄂 Ａ 株的 Ｔ Ｋ 基因存在变异。     

The molecular epidemiology of equine herpesvirus 1 (equine abortion virus) in Australasia 1975 to 1989

The restriction endonuclease DNA fingerprints of 57 isolates of equine herpesvirus 1 (EHV1; equine abortion virus) from abortion, perinatal foal mortalities and encephalitis from 15 epidemics that occurred in Australasia between 1975 and 1989 were examined using the enzymes Bam HI, EcoRI and Bgl II. There was a remarkable degree of uniformity in the restriction patterns; mobility differences were observed in only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located within the repeat structures that bracket the unique short region of the genome or were located at the left terminus of the 150 kilobase pair genome. Based on the Bam HI fingerprints the commonest virus identified in our study was EHV1.IP (P is for prototype strain). There was a single notable exception in that the Bam HI fingerprints of all 8 isolates from one of 3 Victorian farms that experienced abortion in 1989 resembled a variant EHV1.IB that was identified as a cause of abortion in Central Kentucky in 1970 to 1974. We present evidence that EHV1.IB caused abortion in California in 1964 and has remained unaltered in its Bam HI restriction pattern. No antigenic differences were found among 4 distantly related EHV1 isolates, including the variant IB, using a panel of 5 monoclonal antibodies to glycoprotein C (gC), a glycoprotein recognised to be highly variable. The uniformity of these unrelated EHV1 isolates is further evidence for a recent origin for EHV1 and may help to explain the natural history of this virus in the horse in which it seems to be a cause of serious epidemics of abortion and perinatal mortality, and less commonly of encephalitis.     

A human major histocompatibility complex (MHC) DNA probe recognizes goat genes

A human major histocompatibility complex (MHC) class II (DR-β) probe was hybridized with restriction enzyme digest of genomic DNA from 14 goats. Nine of the animals belonged to one family. Digestion of the DNA with the restriction enzyme Eco RI gave 7 fragments in 13 animals and 6 fragments in the last animal. No other polymorphism could be detected. Bam HI digestion gave from 3 to 6 fragments which displayed a considerable polymorphism. In the family studied, polymorphic fragments were inherited together with serologically defined lymphocyte antigen specificities believed to be coded for by MHC class I genes.