表达HSV－2 gD基因的重组痘苗病毒保护小鼠对抗致死量HSV病霉攻击  

RECOMBINANT VIRUS EXPRESSING PCR-MODIED HSV-2gD GENE PROTECT MICE AGAINST LETHAL HSV-2 CHALLENGE

作　　者：容敏清[1] 阎辉[1] 阮力[1] 侯云德[1] 何南祥[1] 朱既明[1] 

机构地区：[1]中国预防医学科学院病毒学研究所,北京病毒基因工程国家重点实验室,浙江医科大学传染病研究所

出　　处：《病毒学报》1995年第3期220-227,共8页Chinese Journal of Virology

基　　金：国家"863"高科技计划生物技术领域资助

摘　　要：利用ＰＣＲ方法对单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）基因进行了修饰，在其５＇端删去约５００ｂｐ的非编码区，仅保留ＡＴＧ上游７个ｂｐ。将修饰后的ＨＳＶ－２ｇＤ基因插入到带有痘苗病毒天坛株ＴＫ基因区段的痘苗表达质粒ｐＪＳＡ１１７５，置于痘苗病毒Ｐ７．５ｋ早／晚期启动子控制下。将此重组质粒用脂质体Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ￣＋痘苗病毒天坛株感染的ＴＫ￣－１４３细胞，通过同源重组机制和标志基因ＬａｃＺ产物的蓝斑显色作用，以及ＢｕｄＲ试剂对ＴＫ表型的选择压力，筛选出整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。Ｓｏｕｔｈｅｍ杂交表明，ＨＳＶ－２ｇＤ基因已正确地插入痘苗病毒ＴＫ基因区内；间接免疫荧光检测显示，ＨＳＶ－２ｇＤ蛋白已得到有效表达，且主要分布于细胞膜。重组病毒免疫家兔可产生明显的抗ＨＳＶ－２ｇＤ中和抗体。用重组病毒免疫小鼠，３周后可使９４％（１７／１８）的小鼠对抗ＨＳＶ－２的致死量攻击，表明重组病毒具有明显的免疫保护作用。he glycoprotein D gene of herpes simplex virus type 2(HSV -2gD )was modified by means of polymerase chain reaction (PCR)to delete about 500bp of its 5'-noncoding region .PCR -modified HSV-2 gD gene was inserted into the plasmid pJSA1175 under the control of P7.5 promoter of vaccinia virus flanked by TK gene sequences of the Tian Tan strain of vaccinia virus. The initiation codon ATG of HSV -2gD was immediate downstream from p7.5 promoter. The recombinant plasmid containing HSV-2gD gene was used, to transfect 143TK￣- cell infected by the wild type TK￣+ vaccinia virus (Tian Tan strain )Afterseveral cycles of selection and screening using the marker lac Z gene together with the selective pressure of BudR for TK￣- phenotype ,recombinant vaccinia virus harboring HSV-2gD gene was picked out and sequentially plaque-purified.Southern blot confirmed that the HSV-2gene had been inserted correctly into the TK region of vaccinia virus genome. Therecombinant virus was shown to be able to express HSV-2 gD gene effectively. Indirect immunofluoresent assay showed the expressed HSV-2gD protein distributed predominantlyon cell membrane. Immunization of rabbits with recombinant virus,a neutralizing antibodyresponseagainst HSV-2 was obtained.Seventeen of 18 mice (17/18,94%)vaccinated with the recmbinant virus were protected from the lethal challenge with HSV-2.

关 键 词：单纯疱疹病毒 疫苗 痘苗病毒载体 基因工程 

分 类 号：R373.9[医药卫生—病原生物学] R392－33[医药卫生—基础医学]