PRV鲁A株的分离鉴定及表达EGFP和CSFV-E2的TK～-重组伪狂犬病毒的构建
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摘要
1．作者从山东疑似猪伪狂犬病发病猪场分离了一株病毒，并对其进行了全面鉴定。该分离毒株在兔肾原代细胞上(RK)上和鸡胚成纤维细胞(CEF)上连传5代均出现典型细胞病变，再接种RK-13细胞、BHK-21细胞、Vero细胞、PK-15细胞和143 TK~-细胞均出现典型细胞病变，在RK-13细胞上的感染滴度为10~(6.52)TCID_(50)／0.1mL；在电镜下可见到典型的伪狂犬病毒粒子；该分离毒对氯仿、乙醚敏感，56℃30min灭活；该分离毒能被伪狂犬病毒标准阳性血清中和；分离毒接种家兔、小白鼠和自然宿主猪均出现典型伪狂犬病症状与病变；提取所分离病毒的DNA为模板，应用特异性引物可以用PCR方法扩增出伪狂犬病毒gD基因中272-534nt之间262bp长的特异性片段。上述结果证明所分离病毒为猪伪狂犬病毒，将其命名为鲁A株(PRV LA Strain)。
2．对PRV“株gD基因进行了克隆和序列测定，表明其ORF为1203bp，可编码400个氨基酸组成的多肽，在整个gD基因的ORF内PRV LA株与PRV Ea株、Hubei株、Rice株，NIA-3株、Kaplan株以及BHV-5、EHV-1、FHV-1、CaHV-1、MDV、HVT、ILTV、HSV-1、HSV-2、SHBV的gD基因比较，核苷酸的同源性分别为98.3％、98.3％、98.0％、98.1％、98.6％以及45.7％、36.8％、36.7％、28.5％、28.4％、25.3％、32.4％、29.3％、31.8％、34.2％，氨基酸的同源性分别为97.8％、97.8％、97.5％、98.1％、98.6％以及34.5％、28.4％、31.8％、31.1％、26.9％、24.5％、19.4％、24.3％、24.7％、25.3％。
发现PRV LA株gD基因与Ea株、Hubei株、Rice株、NIA-3株、Kaplan株的gD基因均在802nt-837nt处有一个C(A)GGCCC重复高变区，其对应的是gD 267位-279位Arg-Pro的重复高变区。正是这一重复高变区的碱基缺失或插入使得PRVgD的ORF在1197nt-1215nt间变化，gD前体的氨基酸残基从398个-404个不等。
用DNAStar对PRV-LA、BHV-5、EHV-1、FHV-1、CaHV-1、MDV-1、HVT、ILTV、HSV-1、HSV-2，SHDV的SD氨基酸所做的进化树表明，它们可以被分成4个特定的组：单纯疱疹病毒组，水痘疱疹病毒组，类马立克氏病病毒组，传染性喉气管炎病毒组。其进化关系可能是单纯疱疹病毒组的gD是由水痘疱疹病毒组进化而来，水痘疱疹病毒组的gD又从类马立克氏病病毒组而来，后者又与ILTV的gD有共同起源，ILTV的gD有其独特的进化途径。因此。疱疹病毒的gD基因在进化上可能起源于ILTV的gD
表丛绿仁荧光士山和拙温汕贞D雍腆蛋山的＊K 刃组伪狂犬9山贞的构注
基因的前体基国。
3．对 PRV LA株比基困进行了克隆和序歹。］jNI］定；表明其 ORF为 963hp，可编码 320
个氨基酸组成勺多肽，在整个比 基因勺ORF 内 PRV LA株与 PRV NIA－3株、PRV Ea
株、叩V洲株、状仁1、伐V6 比基因比较，核有酸白同源性分另为98．9％、99．5％、
99．3＄、36．4％、39．1％，氨基酸的同源性分别为98．4％、99．7y、98．7y、36．6％、37．2％。
PM M株比具有疙疹病毒胸菩激酶催化结构域的保守氨基酸共有序列和亚结构域特
征序列。
前人研究证明痘苗病毒的比基因在进化上可能起源于宿主（鸡）细胞的w基因，
而疤疹病毒比基因的起源一直未明。通过将叩VM孤、人和小鼠的胸替酸激酶、人
脱氧胞旮激酶、人腺菩酸激酶的对应于这两个亚结构域的氨基酸用DN人S tar分析的进
化树表明疙疹病毒的TK与人和小鼠的胸旮酸激酶的亲缘关系比与人脱氧胞喀淀激酶
的亲缘关系更近，刚匕疤疹病毒的TK基因在进化上可能起源于宿主细胞的胸菩酸激
酶基③。
4．提取PRV LA株基因组加A为盯R扩增模板，并根据Genbank己发表的 PRV Kaplan
株叽 区UL25、UL24、UL23、UL22基因序歹J，选择保守序yi］设计了两对11物，这两对
引物分别扩增出位于几23（比）两侧（含部分比基因）的可用于同源重组的左臂片段
（L）和右臂片段（R），L包括部分UL25、全部 UL24、部分比，R包括部分皿及部分
UL22，L和R的拼接片段中比基因内部缺失 270个核菩酸。将L片段和R片段克隆于
nBluescrint M 13－载体上，获得nSKLR；再将绿色荧光蛋白载体pEGFP－CI上含EGFP
及其多克隆位点的完整的基固表达盒插入 PSKLR的 L片段和 R片段之间构成转移载体
PSKLIG；又将猪瘟病毒 l．23Kb的 EZ（SFV—EZ）基因片段插入 PSKLRG的多克隆位点
的 Bgl 11与 PStl之间获得转移载体 PSKLRGEZ。
5．提取 pSKLRG质粒，经单酶切线性化后用脂质体与 PRV Bartha－K61株共转染
143T厂细胞，在细胞培养液中有5一涣脱氧尿喀啃旧rdU）存在的条件下筛选出表达
绿色荧光蛋白的重组 PRV Bartha－K61毒株：PRV rBGFP。
6．提取PSKLRGEZ质粒；经单酶切线性｛匕后用脂质体与＊株叩V共转染BHK－21
细胞，在细胞培养液中含有5’一浚脱氧尿喀喀（BrdU）存在的条件下，经筛选与纯
｛乙获得 T A虫合表达 EGFP与 CSFVEZ 基因的重组病毒北 PRV rGEZ，并对其某些生物学
特性进行了初步鉴定。
7．选门头 5周龄长大二元杂交猪分为 3组，第一组 3头，间隔 10天两次月几注
PRVBartl。a－［61弱毒疫苗各1头份；第＝组6头，间隔10天两次OIL注重组病毒PRV rCEZ
各1?
1. The author isolated and identified one strain of pseudorabies virus (PRV) from a outbreak in a farm in Shandong Province and designated it as PRV LA strain, The isolate was characterized comprehensively. The virus was continually inoculated 5 generations onto rabbit kidney cell and chicken embryonic fibroblast cell (CEF) and induced typical cytopathic effect (CPE).The typical CPE cloud be seen when culturing the virus on RK-13 cell .PK-15cell .BHK-21cell and 143TK~ Cell and the titerof the isolated virus was 10652 TCID50/0.1mL and the typical pseudorabies virus particles could be found under electromicroscope. The isolate was sensitive to chloroform and ethylether and inactivated for 30 minutes at 56癈and could be neuralized by the positive serum to pseudorabies virus. The typical clinical syndrome could be observed when four rabbits and to Balb/c mice and three piglets were infected with the isolate.
The sequence of 262 base pairs lying gD gene could be amplified with the DNA extracted from the isolate and a set of oligonucleotide primers. All of the evidences indicated that the isolated virus was pseudorabies virus.
2. The gD gene of the wild Chinese PRV LA strain was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pGEM-T easy vector and the nucleotides of the gD gene was sequenced. An analysis -of the nucleotide sequence and its deduced amino acid sequence were performed with computer programs. The results revealed that the region of DNA sequenced is 1453 base pairs and it has one ORF of 1203 base pairs to have the potential to encode a protein of 400 amino acids. The gD gene of PRV LA strain shares highly conserved nucleotide with PRV Ea strain, Hubei strain and NIA-3 strain and Kaplan strain. The homology of deduced amino acids of PRV LA strain with PRV Ea strain. Hubei strain and NIA-3 strain and Kaplan strain is 97.8%, 97.8%,97.5%,98.1%,98.6%, respectively. But the PRV gD genes shared limited homology to other alphaherpesviruses equivalents: BHV-5.EHV-1 ,FHV-l,CaHV-l,MDV.HVT,ILTV,HSV-l,HSV-2 and SHBV.
The nucleotide sequence analysis of the 6 PRV strains revealed that there was a highly repeated and altered region which lay to 802nt-837nt in their nucleotide sequences
corresponding to their amino acid sequences 267-279,the insertion and deletion of nucleotides in the region made the gD gene variable between 1197nt-1215nt.
The homology analysis of deduced amino acids of PRV LA strain with other alphaherpesviruses equivalents showed that gD could separate into 4 groups: simplex viruses and varicella viruses and Marek's diseas-like viruses and infectious laryngotracheitis virus and the phlyogenetic tree analysis of the gD sequences may contribute futher evidence of the evolution of gD of alphaherpesviruses from a common ILTV gD progenitor.
3. The thymiding kinase(TK) gene in the unique long region of the wild Chinese PRV LA strain was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pUC 19 plasmid vector and the nucleotides of the TK gene was sequenced. An analysis of the nucleotide sequence and its deduced amino acid sequence were performed with computer programs. The results revealed that the region of DNA sequenced is 1048 base pairs and it has one ORF of 963 base pairs to have the potential to encode a protein of 320 amino acids. The TK gene of PRV LA strain shares highly conserved nucleotide with PRV NIA-3 strain and Ea strain and SH strain. The homology of the nucleotide sequence of PRV LA strain with PRV NIA-3 strain, Ea strain, SH strain, HSV-1, VZV is 98.9%, 99.5%, 99.3%, 36.4% and 39.1%. respectively; and the homology of deduced amino acids among them is 98.4%, 99.7%, 98.7%, 36.6% and 37.2%, respectively. The amino acids of PRV TK contain the conserved ATP-binding motifs (-GXXGXGKT-) and the conserved nucleosite-binding motifs(-DRH-). The similarity of the two conserved bindings between PRV-LA TK and homo sapiens thymidylate kinase and mus musculus thymidylate k
2．对PRV“株gD基因进行了克隆和序列测定，表明其ORF为1203bp，可编码400个氨基酸组成的多肽，在整个gD基因的ORF内PRV LA株与PRV Ea株、Hubei株、Rice株，NIA-3株、Kaplan株以及BHV-5、EHV-1、FHV-1、CaHV-1、MDV、HVT、ILTV、HSV-1、HSV-2、SHBV的gD基因比较，核苷酸的同源性分别为98.3％、98.3％、98.0％、98.1％、98.6％以及45.7％、36.8％、36.7％、28.5％、28.4％、25.3％、32.4％、29.3％、31.8％、34.2％，氨基酸的同源性分别为97.8％、97.8％、97.5％、98.1％、98.6％以及34.5％、28.4％、31.8％、31.1％、26.9％、24.5％、19.4％、24.3％、24.7％、25.3％。
发现PRV LA株gD基因与Ea株、Hubei株、Rice株、NIA-3株、Kaplan株的gD基因均在802nt-837nt处有一个C(A)GGCCC重复高变区，其对应的是gD 267位-279位Arg-Pro的重复高变区。正是这一重复高变区的碱基缺失或插入使得PRVgD的ORF在1197nt-1215nt间变化，gD前体的氨基酸残基从398个-404个不等。
用DNAStar对PRV-LA、BHV-5、EHV-1、FHV-1、CaHV-1、MDV-1、HVT、ILTV、HSV-1、HSV-2，SHDV的SD氨基酸所做的进化树表明，它们可以被分成4个特定的组：单纯疱疹病毒组，水痘疱疹病毒组，类马立克氏病病毒组，传染性喉气管炎病毒组。其进化关系可能是单纯疱疹病毒组的gD是由水痘疱疹病毒组进化而来，水痘疱疹病毒组的gD又从类马立克氏病病毒组而来，后者又与ILTV的gD有共同起源，ILTV的gD有其独特的进化途径。因此。疱疹病毒的gD基因在进化上可能起源于ILTV的gD
表丛绿仁荧光士山和拙温汕贞D雍腆蛋山的＊K 刃组伪狂犬9山贞的构注
基因的前体基国。
3．对 PRV LA株比基困进行了克隆和序歹。］jNI］定；表明其 ORF为 963hp，可编码 320
个氨基酸组成勺多肽，在整个比 基因勺ORF 内 PRV LA株与 PRV NIA－3株、PRV Ea
株、叩V洲株、状仁1、伐V6 比基因比较，核有酸白同源性分另为98．9％、99．5％、
99．3＄、36．4％、39．1％，氨基酸的同源性分别为98．4％、99．7y、98．7y、36．6％、37．2％。
PM M株比具有疙疹病毒胸菩激酶催化结构域的保守氨基酸共有序列和亚结构域特
征序列。
前人研究证明痘苗病毒的比基因在进化上可能起源于宿主（鸡）细胞的w基因，
而疤疹病毒比基因的起源一直未明。通过将叩VM孤、人和小鼠的胸替酸激酶、人
脱氧胞旮激酶、人腺菩酸激酶的对应于这两个亚结构域的氨基酸用DN人S tar分析的进
化树表明疙疹病毒的TK与人和小鼠的胸旮酸激酶的亲缘关系比与人脱氧胞喀淀激酶
的亲缘关系更近，刚匕疤疹病毒的TK基因在进化上可能起源于宿主细胞的胸菩酸激
酶基③。
4．提取PRV LA株基因组加A为盯R扩增模板，并根据Genbank己发表的 PRV Kaplan
株叽 区UL25、UL24、UL23、UL22基因序歹J，选择保守序yi］设计了两对11物，这两对
引物分别扩增出位于几23（比）两侧（含部分比基因）的可用于同源重组的左臂片段
（L）和右臂片段（R），L包括部分UL25、全部 UL24、部分比，R包括部分皿及部分
UL22，L和R的拼接片段中比基因内部缺失 270个核菩酸。将L片段和R片段克隆于
nBluescrint M 13－载体上，获得nSKLR；再将绿色荧光蛋白载体pEGFP－CI上含EGFP
及其多克隆位点的完整的基固表达盒插入 PSKLR的 L片段和 R片段之间构成转移载体
PSKLIG；又将猪瘟病毒 l．23Kb的 EZ（SFV—EZ）基因片段插入 PSKLRG的多克隆位点
的 Bgl 11与 PStl之间获得转移载体 PSKLRGEZ。
5．提取 pSKLRG质粒，经单酶切线性化后用脂质体与 PRV Bartha－K61株共转染
143T厂细胞，在细胞培养液中有5一涣脱氧尿喀啃旧rdU）存在的条件下筛选出表达
绿色荧光蛋白的重组 PRV Bartha－K61毒株：PRV rBGFP。
6．提取PSKLRGEZ质粒；经单酶切线性｛匕后用脂质体与＊株叩V共转染BHK－21
细胞，在细胞培养液中含有5’一浚脱氧尿喀喀（BrdU）存在的条件下，经筛选与纯
｛乙获得 T A虫合表达 EGFP与 CSFVEZ 基因的重组病毒北 PRV rGEZ，并对其某些生物学
特性进行了初步鉴定。
7．选门头 5周龄长大二元杂交猪分为 3组，第一组 3头，间隔 10天两次月几注
PRVBartl。a－［61弱毒疫苗各1头份；第＝组6头，间隔10天两次OIL注重组病毒PRV rCEZ
各1?
1. The author isolated and identified one strain of pseudorabies virus (PRV) from a outbreak in a farm in Shandong Province and designated it as PRV LA strain, The isolate was characterized comprehensively. The virus was continually inoculated 5 generations onto rabbit kidney cell and chicken embryonic fibroblast cell (CEF) and induced typical cytopathic effect (CPE).The typical CPE cloud be seen when culturing the virus on RK-13 cell .PK-15cell .BHK-21cell and 143TK~ Cell and the titerof the isolated virus was 10652 TCID50/0.1mL and the typical pseudorabies virus particles could be found under electromicroscope. The isolate was sensitive to chloroform and ethylether and inactivated for 30 minutes at 56癈and could be neuralized by the positive serum to pseudorabies virus. The typical clinical syndrome could be observed when four rabbits and to Balb/c mice and three piglets were infected with the isolate.
The sequence of 262 base pairs lying gD gene could be amplified with the DNA extracted from the isolate and a set of oligonucleotide primers. All of the evidences indicated that the isolated virus was pseudorabies virus.
2. The gD gene of the wild Chinese PRV LA strain was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pGEM-T easy vector and the nucleotides of the gD gene was sequenced. An analysis -of the nucleotide sequence and its deduced amino acid sequence were performed with computer programs. The results revealed that the region of DNA sequenced is 1453 base pairs and it has one ORF of 1203 base pairs to have the potential to encode a protein of 400 amino acids. The gD gene of PRV LA strain shares highly conserved nucleotide with PRV Ea strain, Hubei strain and NIA-3 strain and Kaplan strain. The homology of deduced amino acids of PRV LA strain with PRV Ea strain. Hubei strain and NIA-3 strain and Kaplan strain is 97.8%, 97.8%,97.5%,98.1%,98.6%, respectively. But the PRV gD genes shared limited homology to other alphaherpesviruses equivalents: BHV-5.EHV-1 ,FHV-l,CaHV-l,MDV.HVT,ILTV,HSV-l,HSV-2 and SHBV.
The nucleotide sequence analysis of the 6 PRV strains revealed that there was a highly repeated and altered region which lay to 802nt-837nt in their nucleotide sequences
corresponding to their amino acid sequences 267-279,the insertion and deletion of nucleotides in the region made the gD gene variable between 1197nt-1215nt.
The homology analysis of deduced amino acids of PRV LA strain with other alphaherpesviruses equivalents showed that gD could separate into 4 groups: simplex viruses and varicella viruses and Marek's diseas-like viruses and infectious laryngotracheitis virus and the phlyogenetic tree analysis of the gD sequences may contribute futher evidence of the evolution of gD of alphaherpesviruses from a common ILTV gD progenitor.
3. The thymiding kinase(TK) gene in the unique long region of the wild Chinese PRV LA strain was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pUC 19 plasmid vector and the nucleotides of the TK gene was sequenced. An analysis of the nucleotide sequence and its deduced amino acid sequence were performed with computer programs. The results revealed that the region of DNA sequenced is 1048 base pairs and it has one ORF of 963 base pairs to have the potential to encode a protein of 320 amino acids. The TK gene of PRV LA strain shares highly conserved nucleotide with PRV NIA-3 strain and Ea strain and SH strain. The homology of the nucleotide sequence of PRV LA strain with PRV NIA-3 strain, Ea strain, SH strain, HSV-1, VZV is 98.9%, 99.5%, 99.3%, 36.4% and 39.1%. respectively; and the homology of deduced amino acids among them is 98.4%, 99.7%, 98.7%, 36.6% and 37.2%, respectively. The amino acids of PRV TK contain the conserved ATP-binding motifs (-GXXGXGKT-) and the conserved nucleosite-binding motifs(-DRH-). The similarity of the two conserved bindings between PRV-LA TK and homo sapiens thymidylate kinase and mus musculus thymidylate k
引文
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[2]陆承平主编，兽医微生物学[M]，第三版，北京，中国农业出版社，2001，411-483．
[3]Thomas C Mettenleiter, PRV: the virus and molecular Pathogenesis[J]. Vet. Res. 2000(31): 99-115.
[4]殷震，刘景华主编，动物病毒学[M]，第二版，北京：科学出版社，1997，998-1220．
[5]娄高明，费恩阁，杜伟贤，PRV分子生物学研究进展[J]。预防兽医学进展，1999，1(4)：9-13．
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[14]Klupp, B. G., Walter Fuchs, Emilie weiland et al. PRV gI is necessary for virus infectivity but dispensable for viron location of gH[J]. J Virol, 1997, 71(10): 7687-7695.
[15]Klupp, B. G., Baumeisfer J., Dietz P. Et., PRV gK is a structural component of virions involved in virus release but not required for entry[J], J Virol.,1998, 72: 1949～1958.
[16]Hanssens, F.P., Nauwgnck H. J., Pensaert M. B Involvement of membrane bound viral glycoproteius in adhesion of PRV infected cells[J]. J Virol, 1993, 67(8): 4492-4496.
[17]Enquist L. W., Husak. P. J., Banfiecd B. W. et al. Infection and spread of alphaherpesviruses in the nervous system[J], Advances in Virus Res., 1999, 51: 237-347.
[18]Rea TJ, Timmins JG, Long GW et al. Mapping and sequence of the gene for the pseudorabies virus glycoprotein which accumulates in the medium of infected cells[J], J Virol. 1985 Apr;54(1):21-9.
[19]Klupp B. G., Jons A.gM and gN of PRV form a disulfide-linked complex[J].J Virol. 1998,72: 550-557.
[20]Klupp BG. Nixdorf R, Mettenleiter TC. Pseudorabies virus glycoprotein M inhibits membrane fusion[J]. J Virol. 2000 Aug: 74 (15) :6760-6768.
[21]Karger A.. Schmidt., Mettenleiter Th. C et al. Infectivity of a pseudorabies virus mutant lacking attachment gC and gD[J], J Virol, 1998, 72: 7314-7348.
[22]Cheung A.K., Jing Fang, Wesley R. D. et al. Characterization of a PRV that is defective in EPO and latency genes[J], Am. J. Vet. Res., 1994, 55: 1710-1716.
[23]Jons A., Gerdts V., Lange E. et al. Attenuation of dUTPase deficient PRV for the natural host[J],Vet Miorobiol, 1997, 56(12): 47-54.
[24]DeWind N., Berns A., Gielkens A. et al. Ribonucleotide reductase-deficient mutants of PRV are avirulent for pigs and induce partial protective immunity[J], J Gen Virol, 1993, 74: 351-359.
[25]Cheung A. K., Cloning of the latency gene and EPO gene of PRV[J], J Virol, 1991, 65: 5260-5271.
[26]Car J.P.. Levitt P., Enquist L. W. et al, Different patterns of neuronal infection after intracerebral injection of two strains of PRV[J], J Virol, 1998, 72: 4434-4441.
[27]王明俊主编，兽医生物制品学[M]，北京，中国农业出版社，2001，603-607．
[28]陈焕春，金梅林，何启盖等，猪伪狂犬病油乳剂灭活疫苗的制备及安全性与免疫性试验[J]，畜牧兽医学报，2001，32(1)，44-51．
[29]魏振明，吴平，陈霖普等，伪狂犬病毒弱毒株若干生物学特性[J]，福建省农科院学报，1995，(10)4：20-25。
[30]S kit, Malon Kit MD, E. C. Pirtle. Attenuated Properties of thymidine kinase negative deletion mutant of Pseudorabies virus[J], Am J Vet Res, Vol 46(6): 1359-1367.
[31]C. C. Marchoili, R. J. Yancey, Jr, R. C. Wardley, et al. A Vaccine strain of Pseudorcibies virus With deletions in the thymidine kinase and glycoprotein X gene[J]. Am J Vet Res, 1987, Vol.48(11) :1577-1583.
[32]J. T. Van Oirschot, R. J. M. Moormann, A. J. M. Berns, et al. Efficiency of a Pseudorabies Virus Vaccine based on deletion mutant Strain 783 that does not express thymidine Kinase and glycoprotein I [J], Am J Vet Res, 1991, Vol 52(7):1056-1060.
[33]王琴，郭万柱，娄高明等，伪狂犬病病毒Fa株胸苷激酶基因缺失株的构建[J]，病毒学报，1996(4)：348-354．
[34]方六荣，周复春，陈焕春等，伪狂犬病病毒鄂A株TK~-／lacZ突变株的构建[J]，畜牧兽医学报，2001，32(3)：244-248．
[35]de wind N, Berm A, Gielkens A, el al. Ribonucleotide reductase -deficient mutants of
Pseudorabies vims are avirulent for pigs and induce partial Protective immunity[J]. .1 Gen virol, 1993, 4:351-359．
[36] Jons A, Gredts V, lange E, el al. Attenuation of dUTPase-deficient. Pseudorabies virus for the natural host[J]. Vet Microbiol, 1997, 56:47-54．
[37] Dijkstra JM, Gerdts V, Klupp BG, et al. Deletion of glycoprotein gM of Pseudorabies Virus
results in attenuation for the natural host[J]. J Gen Virol, 1997,78:2147-2151．
[38] T G Kimman, Niekls De Wind, Tiny De Bruin, et al. Inactivation of glycoprotein gE and tymidine kinase or the US3-encoded protein kinase synergistically decraeses in vivo riplication of pseudorabies virus and the induction of protective immunty[J]. Virol, 1994, 205,511-518．
[39] De Wind N, Peelers BP, Zuderveld A. Gielkens AL. Bems AJ, Kimman TGMutagenesis and characterization of a 41-kilobase-pair region of the pseudorabies virus genome: transcription map, search for viailence genes, and comparison with homologs of herpes simplex virus type 1 [J]. Virology. 1994 May 1 ;200(2) :784-90．
[40] Dean HJ, Cheung AK A deletion at the UL/1R junction reduces pseudorabies virus neurovirulence [J]. J Virol 1995 Feb;69(2) : 1334-8．
[41] S Kit, Michael S, H Ichimura, et al. Second-generention Pseudorabies Virus Vaccine with deletion in thymidine Kinase and glycoprotein genes [J]. Am J Vet Res, 1987,48(5) : 780-793．
[42] Heffner S, Kovacs F, Klupp BG, Mettenleiter TC. Glycoprotein gp50~negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine[J]. J Virol 1993, 7(3) :1529-37．
[43] Rob J. M. Moormann, Tony de Rover, Jan Briaire et al. Inactivation of the thymidine kinase gene of a gl deletion mutant of pseudorabies virus generates a safe but still highly immunogenic vaccine strain[J] .J GEN Virol, 1990, 71:1591-95．
[44] Marchioli C , Kit S, Ichimura H et al. A vaccine strain of pseudorabies virus twith deletion in the thimidine kinase and glycoprotein X gene [J]. Am J Vet Res, 1987,48:780-93．
[45] Messerle. M. I, Crnkovic. W. Hammerschmidt. H. et al. Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome [J]. Proc.Natl.Acad.Sci. USA 1997,94:14759-14763．
[46] Gregory A. Smith, Lynn W. et al .Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus [J]. J Virol, 1999,73:6405-6414．
[47] Henderson LM, Katz J B, Erickson G A, et al. In vivo and in vitro genetic recombination between conventional and gene-deleted vaccine strains of Psendorabie virus[J]. Am J Vet Res, 1990, 51 (10) : 1656-1662．
[48] 仇华吉,童光志,新型伪狂犬病疫苗及其评价[J]。中国兽医杂志,1999,25(7) :34-37。
[49] Hammond JM, Jansen ES, Morrissy CJ, , et al. Vaccination of pigs with a recomhinant porcine
adenovirus expressing the gD gene from pseudorabies virus[J]. Vaccine 2001 , 19(27) :3752-8 .
[50] M. Adam, M. F. Lepottier, M. Eloit. Vaccination of pigs with replication-defective adenovirus vectored vaccines:the example of pseudorabies[J]. Veterrinary Microbiology,1994,42, 205-215．
[51] C. C. Marchioli, R.J. Yancey, JR., E. A. Petrovskis et al.Evalution of pseudorabies virus glycoprotein gp50 as a vaccine for aujeszky's disease in mice and swine:expression hy vaccinia virus and Chinese hamster ovary cells[J]. J Virol 1987, 61 (12) :3977-3982．
[52] Rivere M, Tantaglia J, Perkus M E et al. Protection of mice and swine from Pseudorabies virus conferred by vaccinia virus-based recombinants[J]. J Virol, 1992,66:3424-3434．
[53] Tartaglia J, Perkus M E, Taylor J, et al. .NYVAC:A higly attenuated strain of vaccinia virus[J]. Virol, 1992, 188:217-232．
[54] Brockmieier S L, Lager KM, Mengeling WL. Vaccination with recombinant vaccinia virus vaccines expressing glycoprotein genes of pseudorabies vims in the presence of maternal immuniry[J]. Vet Microbiol 1997 Nov;58(2-4) :93-103．
[55] Van de Leek M .Evalution of swinepox vims as a vaccine vector in pigs using an Aujeszky's diseasevirus gene insert coding forgly coproteins gp50 and gp63[J]. Vet.Rec, 1994,134:13-18．
[56] Kit M.Kit S,Little SP, et al.Bovine herpesvirus-l(infectious bovinerhinotracheitis virus)-based viral vector which expresses foot and mouth disease epitopes[J]. Vaccine, 1991, 9:564-572．
[57] Otsuka H. Xuan X. Construction of BHV-1 recombinants which express PRV gB, gC, gD and gE[J], Arch Virol, 1996, 141: 57-71．
[58] Van Zijl M, Wensvoort G, de Kluyver E,etal. Live attenuated pseudorabies virus expressing envelope glycoprot ion El of hog cholera virus protects swine against pseudorabies and hog cholera[J]. J virol. 1991,65:2761-65．
[59] Peeters B, Bienkowska-szewczykK, Hulst M et al. Biologically safe, non-transmissible pseudorabies vims vector vaccine protects pigs against both aujeszky' s disease and classical swine fever[J]. J Gen virol , 1997(78) :3311-15．
[60] Wolff, JA, Malone KW, Williams, P, et al. Direct gene transfer into mouse muscle in vivo[J]. Science, 1990, 247, 1465-1468．
[61] Volker Gerclts Alice Jons, Birgit Makoschcy. Protection of pigs against Aujeszkys disease by DNA Vaccination[J], J Gen Virol, 1997, 78:2139-2146．
[62] Van Rooij El, Haaginans BL, Glansbeek HL et al. A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs ;ind reduces virus excretion early after infection[J]. Vet Immunol Immunopathol, 2000 ,7-1(1-2) : 121-136．
[63] M. J. Mctfinley, and K. B. plan. Antibody response of pseuclorabies virus subunit-vaccinated pigs to viral nucleocapsid proteins following low-dose virus challenge of immunity[J]. AM J VET RES, Vo150 (8) :1290-93
[2]陆承平主编，兽医微生物学[M]，第三版，北京，中国农业出版社，2001，411-483．
[3]Thomas C Mettenleiter, PRV: the virus and molecular Pathogenesis[J]. Vet. Res. 2000(31): 99-115.
[4]殷震，刘景华主编，动物病毒学[M]，第二版，北京：科学出版社，1997，998-1220．
[5]娄高明，费恩阁，杜伟贤，PRV分子生物学研究进展[J]。预防兽医学进展，1999，1(4)：9-13．
[6]Newcomb W. N., Homa F. L., Thomsen D. R. et al. Assembly of HSV Procapsid from purified components and identification of small complexes containing the major capsid and scaffdding proteins[J],J. Virol, 1999, 73: 4339-4250.
[7]Granzow H., Weiland F., Jons A. et al. Ulstrastructural Analysis of the Replication cycle of PRV in cell Culture: A Reassessment[J],J. Virol, 1997, 71: 2072-2082.
[8]Whealy M.E., Card J. P., Meade R. P. et al. Effect of brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress[J], J. Virol, 1991, 65: 1066-1081.
[9]Brideu A.D., Banfield B. W., Enquist L W., The US9 gene product of PRV is a phosphorylated, tail—anchored type Ⅱ membrane protein[J], J Virol, 1998, 72: 4560-4570.
[10]Cockrell A.S., Muggeridge M. I. Herpes simplex virus 2 UL45 is a type Ⅱ membrane protein [J].J Virol, 1998, 72:4430-4433.
[11]Peeters B., De Wind N., Minetta Hooisma et al. PRV gp50 and gⅡ are essential for virus penetration., but only gⅡ is involved in membrane fusion [J]. J Virol, 1992, 66(2): 894-965.
[12]Geraghty R.J., Krummenacher C., Cohen G. H. et al. Entry of alphaherpesvirus mediated by poliovirus receptor—related Protein 1 and poliovirus receptor[J], Science, 1998, 280: 1618-1620.
[13]Babic. N., Klupp B. G., Makoschey. B et al. PRV gH is essential for penetration and propagation in cell cultcme and in the nervous system of mice[J], J Gen Virol, 1996, 77: 2277-2285.
[14]Klupp, B. G., Walter Fuchs, Emilie weiland et al. PRV gI is necessary for virus infectivity but dispensable for viron location of gH[J]. J Virol, 1997, 71(10): 7687-7695.
[15]Klupp, B. G., Baumeisfer J., Dietz P. Et., PRV gK is a structural component of virions involved in virus release but not required for entry[J], J Virol.,1998, 72: 1949～1958.
[16]Hanssens, F.P., Nauwgnck H. J., Pensaert M. B Involvement of membrane bound viral glycoproteius in adhesion of PRV infected cells[J]. J Virol, 1993, 67(8): 4492-4496.
[17]Enquist L. W., Husak. P. J., Banfiecd B. W. et al. Infection and spread of alphaherpesviruses in the nervous system[J], Advances in Virus Res., 1999, 51: 237-347.
[18]Rea TJ, Timmins JG, Long GW et al. Mapping and sequence of the gene for the pseudorabies virus glycoprotein which accumulates in the medium of infected cells[J], J Virol. 1985 Apr;54(1):21-9.
[19]Klupp B. G., Jons A.gM and gN of PRV form a disulfide-linked complex[J].J Virol. 1998,72: 550-557.
[20]Klupp BG. Nixdorf R, Mettenleiter TC. Pseudorabies virus glycoprotein M inhibits membrane fusion[J]. J Virol. 2000 Aug: 74 (15) :6760-6768.
[21]Karger A.. Schmidt., Mettenleiter Th. C et al. Infectivity of a pseudorabies virus mutant lacking attachment gC and gD[J], J Virol, 1998, 72: 7314-7348.
[22]Cheung A.K., Jing Fang, Wesley R. D. et al. Characterization of a PRV that is defective in EPO and latency genes[J], Am. J. Vet. Res., 1994, 55: 1710-1716.
[23]Jons A., Gerdts V., Lange E. et al. Attenuation of dUTPase deficient PRV for the natural host[J],Vet Miorobiol, 1997, 56(12): 47-54.
[24]DeWind N., Berns A., Gielkens A. et al. Ribonucleotide reductase-deficient mutants of PRV are avirulent for pigs and induce partial protective immunity[J], J Gen Virol, 1993, 74: 351-359.
[25]Cheung A. K., Cloning of the latency gene and EPO gene of PRV[J], J Virol, 1991, 65: 5260-5271.
[26]Car J.P.. Levitt P., Enquist L. W. et al, Different patterns of neuronal infection after intracerebral injection of two strains of PRV[J], J Virol, 1998, 72: 4434-4441.
[27]王明俊主编，兽医生物制品学[M]，北京，中国农业出版社，2001，603-607．
[28]陈焕春，金梅林，何启盖等，猪伪狂犬病油乳剂灭活疫苗的制备及安全性与免疫性试验[J]，畜牧兽医学报，2001，32(1)，44-51．
[29]魏振明，吴平，陈霖普等，伪狂犬病毒弱毒株若干生物学特性[J]，福建省农科院学报，1995，(10)4：20-25。
[30]S kit, Malon Kit MD, E. C. Pirtle. Attenuated Properties of thymidine kinase negative deletion mutant of Pseudorabies virus[J], Am J Vet Res, Vol 46(6): 1359-1367.
[31]C. C. Marchoili, R. J. Yancey, Jr, R. C. Wardley, et al. A Vaccine strain of Pseudorcibies virus With deletions in the thymidine kinase and glycoprotein X gene[J]. Am J Vet Res, 1987, Vol.48(11) :1577-1583.
[32]J. T. Van Oirschot, R. J. M. Moormann, A. J. M. Berns, et al. Efficiency of a Pseudorabies Virus Vaccine based on deletion mutant Strain 783 that does not express thymidine Kinase and glycoprotein I [J], Am J Vet Res, 1991, Vol 52(7):1056-1060.
[33]王琴，郭万柱，娄高明等，伪狂犬病病毒Fa株胸苷激酶基因缺失株的构建[J]，病毒学报，1996(4)：348-354．
[34]方六荣，周复春，陈焕春等，伪狂犬病病毒鄂A株TK~-／lacZ突变株的构建[J]，畜牧兽医学报，2001，32(3)：244-248．
[35]de wind N, Berm A, Gielkens A, el al. Ribonucleotide reductase -deficient mutants of
Pseudorabies vims are avirulent for pigs and induce partial Protective immunity[J]. .1 Gen virol, 1993, 4:351-359．
[36] Jons A, Gredts V, lange E, el al. Attenuation of dUTPase-deficient. Pseudorabies virus for the natural host[J]. Vet Microbiol, 1997, 56:47-54．
[37] Dijkstra JM, Gerdts V, Klupp BG, et al. Deletion of glycoprotein gM of Pseudorabies Virus
results in attenuation for the natural host[J]. J Gen Virol, 1997,78:2147-2151．
[38] T G Kimman, Niekls De Wind, Tiny De Bruin, et al. Inactivation of glycoprotein gE and tymidine kinase or the US3-encoded protein kinase synergistically decraeses in vivo riplication of pseudorabies virus and the induction of protective immunty[J]. Virol, 1994, 205,511-518．
[39] De Wind N, Peelers BP, Zuderveld A. Gielkens AL. Bems AJ, Kimman TGMutagenesis and characterization of a 41-kilobase-pair region of the pseudorabies virus genome: transcription map, search for viailence genes, and comparison with homologs of herpes simplex virus type 1 [J]. Virology. 1994 May 1 ;200(2) :784-90．
[40] Dean HJ, Cheung AK A deletion at the UL/1R junction reduces pseudorabies virus neurovirulence [J]. J Virol 1995 Feb;69(2) : 1334-8．
[41] S Kit, Michael S, H Ichimura, et al. Second-generention Pseudorabies Virus Vaccine with deletion in thymidine Kinase and glycoprotein genes [J]. Am J Vet Res, 1987,48(5) : 780-793．
[42] Heffner S, Kovacs F, Klupp BG, Mettenleiter TC. Glycoprotein gp50~negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine[J]. J Virol 1993, 7(3) :1529-37．
[43] Rob J. M. Moormann, Tony de Rover, Jan Briaire et al. Inactivation of the thymidine kinase gene of a gl deletion mutant of pseudorabies virus generates a safe but still highly immunogenic vaccine strain[J] .J GEN Virol, 1990, 71:1591-95．
[44] Marchioli C , Kit S, Ichimura H et al. A vaccine strain of pseudorabies virus twith deletion in the thimidine kinase and glycoprotein X gene [J]. Am J Vet Res, 1987,48:780-93．
[45] Messerle. M. I, Crnkovic. W. Hammerschmidt. H. et al. Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome [J]. Proc.Natl.Acad.Sci. USA 1997,94:14759-14763．
[46] Gregory A. Smith, Lynn W. et al .Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus [J]. J Virol, 1999,73:6405-6414．
[47] Henderson LM, Katz J B, Erickson G A, et al. In vivo and in vitro genetic recombination between conventional and gene-deleted vaccine strains of Psendorabie virus[J]. Am J Vet Res, 1990, 51 (10) : 1656-1662．
[48] 仇华吉,童光志,新型伪狂犬病疫苗及其评价[J]。中国兽医杂志,1999,25(7) :34-37。
[49] Hammond JM, Jansen ES, Morrissy CJ, , et al. Vaccination of pigs with a recomhinant porcine
adenovirus expressing the gD gene from pseudorabies virus[J]. Vaccine 2001 , 19(27) :3752-8 .
[50] M. Adam, M. F. Lepottier, M. Eloit. Vaccination of pigs with replication-defective adenovirus vectored vaccines:the example of pseudorabies[J]. Veterrinary Microbiology,1994,42, 205-215．
[51] C. C. Marchioli, R.J. Yancey, JR., E. A. Petrovskis et al.Evalution of pseudorabies virus glycoprotein gp50 as a vaccine for aujeszky's disease in mice and swine:expression hy vaccinia virus and Chinese hamster ovary cells[J]. J Virol 1987, 61 (12) :3977-3982．
[52] Rivere M, Tantaglia J, Perkus M E et al. Protection of mice and swine from Pseudorabies virus conferred by vaccinia virus-based recombinants[J]. J Virol, 1992,66:3424-3434．
[53] Tartaglia J, Perkus M E, Taylor J, et al. .NYVAC:A higly attenuated strain of vaccinia virus[J]. Virol, 1992, 188:217-232．
[54] Brockmieier S L, Lager KM, Mengeling WL. Vaccination with recombinant vaccinia virus vaccines expressing glycoprotein genes of pseudorabies vims in the presence of maternal immuniry[J]. Vet Microbiol 1997 Nov;58(2-4) :93-103．
[55] Van de Leek M .Evalution of swinepox vims as a vaccine vector in pigs using an Aujeszky's diseasevirus gene insert coding forgly coproteins gp50 and gp63[J]. Vet.Rec, 1994,134:13-18．
[56] Kit M.Kit S,Little SP, et al.Bovine herpesvirus-l(infectious bovinerhinotracheitis virus)-based viral vector which expresses foot and mouth disease epitopes[J]. Vaccine, 1991, 9:564-572．
[57] Otsuka H. Xuan X. Construction of BHV-1 recombinants which express PRV gB, gC, gD and gE[J], Arch Virol, 1996, 141: 57-71．
[58] Van Zijl M, Wensvoort G, de Kluyver E,etal. Live attenuated pseudorabies virus expressing envelope glycoprot ion El of hog cholera virus protects swine against pseudorabies and hog cholera[J]. J virol. 1991,65:2761-65．
[59] Peeters B, Bienkowska-szewczykK, Hulst M et al. Biologically safe, non-transmissible pseudorabies vims vector vaccine protects pigs against both aujeszky' s disease and classical swine fever[J]. J Gen virol , 1997(78) :3311-15．
[60] Wolff, JA, Malone KW, Williams, P, et al. Direct gene transfer into mouse muscle in vivo[J]. Science, 1990, 247, 1465-1468．
[61] Volker Gerclts Alice Jons, Birgit Makoschcy. Protection of pigs against Aujeszkys disease by DNA Vaccination[J], J Gen Virol, 1997, 78:2139-2146．
[62] Van Rooij El, Haaginans BL, Glansbeek HL et al. A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs ;ind reduces virus excretion early after infection[J]. Vet Immunol Immunopathol, 2000 ,7-1(1-2) : 121-136．
[63] M. J. Mctfinley, and K. B. plan. Antibody response of pseuclorabies virus subunit-vaccinated pigs to viral nucleocapsid proteins following low-dose virus challenge of immunity[J]. AM J VET RES, Vo150 (8) :1290-93