单纯疱疹病毒I型糖蛋白D核酸疫苗的构建及初步研究  

Construction of Herpes Simplex Virus TypeⅠGlycoprotein D DNA Vaccine and Its Preliminary Study

作　　者：朱明昭[1] 刘宏伟[1] 刘晓娟[1] 韩晔华[1] 杨宝玲[1] 宋国兴 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所生物物理室,北京100005

出　　处：《中国医学科学院学报》2002年第1期67-70,共4页Acta Academiae Medicinae Sinicae

摘　　要：目的构建、制备Ⅰ型单纯疱疹病毒糖蛋白Ｄ重组质粒ＤＮＡ疫苗，初步检测其诱导机体产生体液免疫应答的效果，为研制ＨＳＶ－１新型疫苗奠定基础。方法用ＰＣＲ的方法从ＨＳＶ－１病毒基因组中扩增糖蛋白Ｄ（ｇｌｙｃｏｐｒｏｔｅｉｎ　Ｄ，ｇＤ）基因，利用基因重组技术构建重组质粒；将重组质粒体外转染真核细胞ＣＯＳ－７，Ｗｅｓｔｅｒｎ　ｂｌｏｔｔｉｎｇ检测表达产物；于ＢＡＬＢ／Ｃ小鼠后腿胫前肌注射免疫，０、２周各免疫１次，１００μｇ／次。初次免疫后０、２、４、６周眼眶采血，ＥＬＩＳＡ间接法检测抗体。结果重组质粒酶切出相应大小片段，经测序证实为ＨＳＶ－１ｇＤ序列。Ｗｅｓｔｅｒｎ　ｂｌｏｔｔｉｎｇ证实能够在体外真核细胞中表达。免疫小鼠后，产生特异性抗体，抗体滴度１∶２０００。结论ＨＳＶ－１ｇＤ重组质粒ＤＮＡ有可能作为ＨＳＶ－１的ＤＮＡ疫苗，用于防治ＨＳＶ－１感染及其相关疾病。Objective The goal of this study was to construct a eukaryotic expression plasmid containing the gene encoding herpes simplex virus typeⅠglycoprotein D (HSV-1,gD)and evaluate its utility for DNA immunization in mice.Methods The gD gene was amplified from viral DNA using PCR with EcoRⅠand BamHⅠrestriction sites encoded on5'and3'ends,respectively.The PCR fragment was inserted into the transfer vector pGEM-T Easy.gD was then cut from this vector and inserted into the EcoRⅠand BamHⅠsites in the pcDNA3.1at the multiple cloning sites (MCS).The recombinant plasmid,pcDNA3.1-gD1,was transfected into COS-7cells using Lipofectamine according to the manufacture's instructions.The expression of the glycoprotein D was analyzed by immunoblotting of the cell lysates.4-6weeks old BALB/C mice were given two injections at tibia anterialis muscle,each containing100μg of plasmid DNA,on days0and15.pcDNA3.1was used as negative control.Blood samples were taken from all mice at weeks0,2,4,and6after the first inoculation.Standard indirect ELISA was employed to evaluate the levels of specific total Ig in serum.Results The recombinant plasmid was confirmed with restriction digestion and sequencing to contain target gene segment and expressed in COS-7cells in vitro shown by Western blotting.The pcDNA3.1-gD1immunized group induced specific antibody response as compared to the negative control,and the titer was about 1∶2000.Conclusions The recombinant plasmid pcDNA3.1-gD1is potential to be used as a candidate vaccine,for the treatment of HSV-1infection.

关 键 词：单纯疱疹病毒 角膜炎 DNA疫苗 糖蛋白D 构建 

分 类 号：R772.2[医药卫生—眼科] Q786[医药卫生—临床医学]