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.     

鸡传染性喉气管炎病毒gB基因的扩增及酶切分析

以从内蒙古区分离的2株传染性喉气管炎病毒(ILTV)株的DNA为模板，用设计好的1对引物对这2株毒株的gB基因进行了PCR扩增，并对扩增产物分别用BglⅡ、PstⅠ、HindⅢ限制性内切酶酶切分析。结果表明，2个分离株均能特异性地扩增出该病毒gB基因片段，片段大小完全一致，为2．6kb；扩增产物的酶切图谱与参考株完全一致。     

鸭肠炎病毒PCR产物的克隆及鉴定

根据已发达的鸭肠炎病毒（DEV）基因的核苷酸序列，设计并合成了1对引物，以鸭肠炎病毒DNA为模板扩增出602bp的基因片段，将该基因片段通过粘端连接克隆到质粒pBluescipt ⅡKS^ 中，重组质粒转化大肠杆菌DH5α感受态细胸质，在LB平板上筛选重组菌，重组子经聚合酶链反应（PCR）和HindⅢ与PstⅠ双酶切鉴定，该基因片段已成功的克隆到质粒载体上。     

腐蹄病C型节瘤拟杆菌纤毛蛋白基因的克隆与表达载体的构建

用PCR技术从腐蹄病C型节瘤拟杆菌克隆出具有免疫保护性抗原0.85kb纤毛蛋白基因（pili基因），利用该基因构建了纤毛蛋白基因表达载体。提取C型节瘤拟杆菌染色体DNA；用所设计的专一性引物进行PCR，扩增出pili基因；将pili基因克隆于TE载体，TE－pili重组质粒pili基因序列测定结果正确，用EcoRI酶切，低熔点胶回收pili基因片段，经klenow补平后，用T4DNA连接酶将其与中间载体pPLλ连接，将pili基因克隆于pPLλ载体，经BamHI、BamHI HindⅢ、Dral酶切鉴定pili基因正向插入pPLλ载体；扩增pPLλ-pili重组质粒，用BamHI酶切出2.1kb大小片段，回收后，与PME290表达质粒连接，转化宿主细胞PAK／2pfs中，对获得的重组质粒用BamHI酶切，出现2.1kb大小的带,证明含有目的基因。     

犬瘟热病毒GZ-Z株H基因原核表达载体的构建

利用实验室构建的含有犬瘟热病毒H基因的p MD18-H质粒,根据其序列设计带有Bam HⅠ和HindⅢ酶切位点的引物,对H基因ORF进行PCR扩增,得到约1 949 bp的片段。将该片段克隆到p MD18-T载体内,用Bam HⅠ和HindⅢ进行双酶切鉴定、质粒PCR鉴定,筛选出阳性克隆。将阳性克隆再用Bam HⅠ和HindⅢ进行双酶切,纯化回收CDV H基因ORF片段;将原核表达载体p ET32a(+)用同样的方法酶切,回收载体片段,并用T4连接酶将以上两回收片段连接,构建原核表达载体质粒p ET32-H,后经Bam HⅠ和HindⅢ双酶切、质粒PCR、测序进行系列鉴定。结果表明,p ET32-H原核表达质粒构建成功,其中插入片段大小为1 842 bp,可编码607个氨基酸残基。该实验为下一步H蛋白的原核表达及单克隆抗体的制备奠定了基础。     

鸡传染性喉气管炎病毒中国王岗株gX基因的克隆及鉴定

以ｐＵＣ１９质粒为载体克隆鸡传染性喉气管炎病毒（ＩＬＴＶ）中国王岗株的ＤＮＡ，构建了鸡传染性喉气管炎病毒ＤＮＡ的ＫｐｎⅠＤＮＡ文库。参考ＩＬＴＶ－ＳＡ２株ｇＸ基因的核酸序列，设计并合成了分别为１２ｂｐ和１３ｂｐ的１对引物。以ＩＬＴＶ中国王岗株ＤＮＡ为模板，用ＰＣＲ方法特异性地扩增出０．８４ｋｂ的ＩＬＴＶ－ｇＸ基因片段。以地高辛标记该０．８４ｋｂ的片段为探针，经Ｓｏｕｔｈｅｒｎ杂交从ＩＬＴＶ中国王岗株ＫｐｎⅠＤＮＡ文库中筛选出３个含５．２ｋｂ外源ＩＬＴＶＤＮＡ片段的ｇＸ基因阳性重组子。经酶切分析、Ｓｏｕｔｈｅｒｎ杂交、ＰＣＲ检测和该片段部分酶谱分析表明，ＩＬＴＶ中国王岗株ＤＮＡ５．２ｋｂ的ＫｐｎⅠ片段无论是片段大小还是酶切图谱均与ＩＬＴＶ－ＳＡ２株完全相同，而且Ｓｏｕｔｈｅｒｎ杂交和ＰＣＲ检测均为ｇＸ阳性，证明其中含有完整的ｇＸ基因     

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。     

旋毛虫新生幼虫cDNA文库构建及其特异性基因的筛选与鉴定

构建旋毛虫新生幼虫cDNA文库并对旋毛虫新牛幼虫期特异件基因进行筛选与鉴定,筛选出期特异性的基因片段.用λZAPⅡExpress载体成功地构建了旋毛虫新生幼虫的cDNA文库,并用放射性同位素a-38P标记cDNA探针对筛选出的阳性克隆进行鉴定.所构建的cDNA文库容量为1.92×106,重组效率为98.6%,所有的克隆片段都在0.5～2.0 kb之间,筛选获得7个阳性克隆,其大小在1.4 kb左右;用放射性同位素a-32P标记cDNA探针后,与旋毛虫成虫、肌幼虫、新生幼虫及成虫/新生幼虫cDNA文库质粒DNA进行Southern印迹杂交,结果与新生幼虫和成虫、新生幼虫cDNA文库质粒DNA有杂交而与成虫、肌幼虫cDNA文库质粒DNA不杂交.该基因是新生幼虫期所特有的cDNA片段.     

Identification of the thymidine kinase gene of infectious laryngotracheitis virus.

An an initial step in the development of a recombinant poultry infectious laryngotracheitis virus (ILTV) vaccine, we report on the identification, cloning, and sequencing of a thymidine kinase (tk) gene from a virulent U.S. field isolate of ILTV. Degenerate oligonucleotide primers for the consensus nucleotide (ATP) binding site and the nucleoside (thymidine) binding site of other herpesvirus tk genes were used in the polymerase chain reaction (PCR) to amplify a fragment of ILTV DNA. The 344-base-pair (bp) amplified fragment was cloned into plasmid pKSII and used in Southern hybridizations to locate the ILTV tk gene on a 2.4-kb HindIII fragment. Upon cloning and sequencing this fragment, a 1089-bp open reading frame was identified, which is predicted to encode a protein demonstrating 27.9% amino acid homology to the herpes simplex virus type 1 (HSV-1) thymidine kinase protein. Analysis of the sequence revealed one region of difference from that reported for the Thorne strain of ILTV. In addition, the portion of the TK protein corresponding to the nucleotide binding domain is highly conserved among the avian herpesviruses.     

PCR扩增TK基因检测鸡传染性喉气管炎病毒的研究

根据已发表序列设计一对包含鸡传染性喉气管炎病毒（ILTV）TK基因全长核苷酸的1259bp引物，对2株ILTV强毒和1株ILTV疫苗毒进行PCR扩增，均能扩增出预期大小的目的片段，酶切分析证实了目的片段的特异性，而其它禽病原体的扩增均为阴性。PCR检测ILTV DNA的最小检测量为75pg。此方法检测人工接种鸡的气管棉拭样品，均能检测到ILTV，因此可用于临诊样品鸡传染性喉气管炎病的检测和诊断。     

应用PCR检测鸡传染性喉气管炎病毒

根据传染性喉气管炎病毒(ILTV)TK基因序列，设计、合成1对引物，应用PCR技术对ILTV以色列疫苗株、河南分离株（ILTV-CG和ILTV-XY）进行PCR扩增，均能扩增出预期大小的目的片段，测序分析和酶切分析证实了PCR产物的特异性，而对其它禽病原体的扩增均为阴性。PCR检测ILTV DNA的最小检测量为21 pg。应用PCR检测人工接种后不同〖JP2〗天数采集的鸡的结膜拭子，接种后第2~5 d均能检测到ILTV。该方法可用于鸡传染性喉气管炎病的诊断和临诊样品检测。     

鸡传染性喉气管炎病毒PCR诊断方法的建立与应用

根据GenBank登录的传染性喉气管炎病毒(ILTV)的TK基因序列设计并合成1对特异性引物,以ILTV疫苗株DNA为模板,建立了检测ILTV TK基因的PCR方法。应用该方法能从临床分离毒株和疫苗株中扩增到长为427 bp的目的片段;但不能从新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)、禽呼肠孤病毒(ARV)、减蛋综合征病毒(EDSV)、H9亚型禽流感病毒(H9-AIV)、传染性支气管炎病毒(IBV)、大肠杆菌以及金黄色葡萄球菌等病原中扩增出阳性条带;敏感性试验表明其DNA最小检出量为4.9 ng;应用该方法和病毒分离法对2份临床病例和人工感染鸡的检测,两者符合率为100%。上述结果表明该PCR方法具有良好的特异性和敏感性,可用于传染性喉气管炎病毒鉴定和临床诊断。     

鸡传染性喉气管炎病毒的分离和TK基因鉴定

从临床表现以呼吸困难、喉头出血为主要特征的某蛋鸡场患鸡喉头和气管等组织分离到一株病毒。该分离毒无血凝特性,应用检测ILTVTK基因的PCR能从分离毒中扩增出大小为427bp的特异性目的片段。测序结果与GenBank收录的ILTV不同毒株序列的同源性为100%。结果初步表明该分离毒为鸡传染性喉气管炎病毒(命名为ILTV-FJ)。     

鸡传染性喉气管炎病毒王岗株gC和gD基因的克隆及序列分析

根据已发表的传染性喉气管炎病毒（ＩＬＴＶ）ＳＡ－２株的核酸序列,设计了１对引物,以ＩＬＴＶ－中国王岗株ＤＮＡ为模板,ＰＣＲ法扩增出１条１．３９ｋｂ的基因片段,将扩增产物插入到真核表达质粒ｐＣＲ＾ＴＭ３－Ｕｎｉ,得到重组质粒ｐＴＡ－ｇＤ,另将克隆到ｐＢｌｕｅｓｃｒｉｐｔ ＳＫ质料中的ｇＣ基因酶毁后,插入到ｐＣＲ＾ＴＭ３－Ｕｎｉ载体,得到重组质粒ｐＴＡ－ｇＣ,经电泳分析、酶分、ＰＣＲ鉴定后,进行序列测     

伪狂犬病病毒HS株tk基因的PCR扩增与克隆

参照伪狂犬病病毒（Ｐｓｅｕｄｏｒａｂｉｅｓｖｉｒｕｓ,ＰＲＶ）ＮＩＡ－３株ｔｋ基因序列,设计并合成１对长２２ｍｅｒ的引物,引物间距１．５ｋｂ,其内包含完整的ＰＲＶｔｋ基因。以ＢＨＫ２１细胞繁殖的ＰＲＶ湖北地方强毒株（ＨＳ－９３０４）基因组为模板进行ＰＣＲ扩增。琼脂糖凝胶电泳检测显示扩增主带清晰,长约１．５ｋｂ,符合设计要求。扩增片段克隆至由ｐＵＣ１８质粒改制而成的Ｔ载体中,限制性内切酶ＢａｍＨＩ、ＳｍａⅠ、ＸｈｏⅠ、ＨｉｎｄⅢ酶切分析证实,扩增片段的酶切位点与ｔｋ基因一致,说明扩增和克隆片段包含ＰＲＶｔｋ基因。     

Analysis of Korean strains of infectious laryngotracheitis virus by nucleotide sequences and restriction fragment length polymorphism

Korean field strains of infectious laryngotracheitis virus (ILTV) were analyzed by comparison of nucleotide sequences of thymidine kinase (TK) and glycoprotein G (gG) genes and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns. Main differences among TK gene sequence were found in both amino acid at 252 and mRNA polyadenylation signals. In virulent strains, amino acid 252 of TK gene was methionine but was threonine in low virulence and vaccine strains. The mRNA polyadenylation signals of TK gene were identified at 24bp downstream from the stop codon in virulent strains, but not in low virulence and vaccine strains. The gG gene of all virulent strains showed the same nucleotide sequence except for N87278 which had a gG gene sequence identical to that of vaccine strains. The virulent ILTV strains differed from low virulence and vaccine strains in PCR-RFLP patterns of TK and gG genes. The RFLP patterns of TK and gG genes of low virulence ILTV strains were identical to those of vaccine strains. In the case of N87278, the PCR-RFLP patterns of TK and gG genes were identical to those of virulent and vaccine strains of ILTV, respectively. From these results, ILTV field strains were classified into three groups according to sequences of TK and gG genes and PCR-RFLP, and the virulent ILTV strains could be discriminated from low virulence and vaccine strains by PCR-RFLP of TK gene. And it was suspected that N87278 might be produced by in vivo recombination between virulent and vaccine strains of ILTV.     

Efficacy of live virus vaccines against infectious laryngotracheitis assessed by polymerase chain reaction-restriction fragment length polymorphism

The efficacy of four different commercial live vaccines (vaccines A, B, C, and D) against the infectious laryngotracheitis virus (ILTV) was assessed in specific-pathogen-free (SPF) chickens. SPF chickens were vaccinated intraocularly at 6 wk old with ILTV live vaccines and were challenged intratracheally with the N91B01 strain of virulent Korean ILTV 2 wk after vaccination. The immunity against ILTV live vaccines was assessed by the incidence of latent infection by the challenge virus in the chickens' tracheas and trigeminal ganglia, the reisolation rate of the challenge virus, and the clinical signs in the chickens challenged with the N91B01 strain of ILTV. The latent infection in chickens was assessed by nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our data showed that the clinical signs and challenge virus isolation were negative in all chickens receiving four difference commercial ILTV live vaccines. The viral DNA of the vaccine strain, but not that of the challenge virus, was detected in chickens vaccinated with vaccine A by nested PCR-RFLP. The viral DNAs of both the vaccine and challenge strains were detected from chickens vaccinated with vaccines B, C, and D. This study showed that only vaccine A can protect chickens from latent infection with the field virulent ILTV. We speculate that the efficacy of infectious laryngotracheitis live vaccines to protect chickens from latent infection with virulent ILTVs can be assessed by nested PCR-RFLP analysis.     

传染性喉气管炎病毒(ILTV)糖蛋白gB在重组鸡痘病毒中的表达

本试验首先用ＰＣＲ方法扩增出ＩＬＴＶ ｇＢ基因,将其克隆到质粒ｐＵＣ１１９中,并进行了序列分析。将克隆的ｇＢ基因再亚克隆到禽痘病毒转移载体质粒中,然后,通过质脂体方法转染由禽痘病毒感染的鸡胚成纤维细胞（ＣＥＦ）中,筛选蓝色的重组病毒,并对其进行了６轮蚀斑纯化。该重组病毒通过ＰＣＲ方法鉴定证明其基因组中含有完整的鸡传染性喉气管炎ｇＢ基因,Ｗｅｓｔｅｒｎ ｂｌｏｔ实验证明了该重组鸡痘病毒表达了鸡传染性喉气管炎ｇＢ糖蛋白。