伪狂犬病病毒糖蛋白gp50基因的克隆和表达  

Cloning and Expression of the gp50 Gene of Pseudorabies Viurus

作　　者：娄高明[1] 郭万柱[1] 韩素文[1] 张宏权[1] 王新平[1] 费恩阁[1] 

机构地区：[1]解放军农牧大学生物工程室,四川农业大学动物科技学院,北京军事医学科学院八所

出　　处：《中国兽医学报》1996年第3期226-233,共8页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金

摘　　要：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。The Bam HI-7 fragment of the PRV Min-A strain was first recovered af-ter the digestion of the recombinant plasmid pPR128 with Bam HI;secondly 2.l kbfragn1eiit was isolated after the llam Hl7 fragment digested with Bst XI,which con-tained complete gp50 gene except 50 bp before ATG of gp50 gene and 890 bp after TAGof gp50 gene;thirdly 2.1kb fragment after digestion with Kpn I and Stu I was in-serted into the plasmid pUC19, the recombinant plasmids of pBK6,pKK4,pKE36 andpSE5 for sequencing 2.1 kb fragment were constructed.The sequence analysis of thegp50 by Sanger's didexoxy termination method showed it to be the same as previouslyreported.The results above indicated that the gp50 gene cloned is correct.The 2.1 kband 1.6 kb fragments that contained complete gp50 gene were inserted into the plasmidpGJP-5 under the control of 7.5kDa protein promoter of vaccinia virus flanked by TKgene sequences of the Tian Tan strain of vaccinia virus.Both chimeric plasmids pG-BT50-36 (generated from 2.1 kb fragment) and pGBT50-S2 (generated from 1.6 kbfragment)comtaining TK flanking sequence were constructed.The plasmids pGBT50-36 and pGBT50-S2 were transferred into CV-1 cells or human 143 TK cells which werepreviously infected by the wild type TK+vaccinia virus(Tian Tan 761 strain).Homolo-gous recombination between plasmids and wildtype virus DNA made gp50 gene integrateinto virus genome in vivo.Recombinant viruses showing TK phenotype were screenedunder 5-BdUR selection pressure. Southern blot hybridization comfirmed that the PRVgp50 gene had been inserted correctly into the TK region of vaccinia virus genome.Re-combinant viruses were used to infect human TK143 cells,and the recombinant viruseswere detected for gp 50 antigenicity by ELISA.The results showed that three recombi-nant vaccinia viruses could express gp50 gene.The gp50 expression level of the recombi-nant viruses V-50B and V-50C (generated from pGBT50-S2)were slightly higher thanthat of the recombination vaccinia virus V-50A (generated from pGBT50-36).

关 键 词：伪狂犬病 病毒 gp50 重组痘苗病毒 克隆 表达 

分 类 号：S852.65[农业科学—基础兽医学]