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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。关键词单纯疱疹病毒角膜炎ＤＮＡ疫苗糖蛋白Ｄ
Objective To construct and prepare the recombinant plasmid DNA vaccine of herpes simplex virus type 1 glycoprotein D and preliminarily test its effect on inducing humoral immune response in vivo to lay the foundation for the development of a new HSV-1 vaccine. Methods The glycoprotein D (gD) gene was amplified from the genome of HSV-1 virus by PCR, and the recombinant plasmid was constructed by gene recombination technique. The recombinant plasmid was transfected into COS-7 cells in vitro and detected by Western blotting The BALB / C mice were injected immunized with the anterior tibialis anterior muscle, and each immunized for 1 week and 2 weeks with 100 μg / time. Orbital blood sampling at 0, 2, 4, and 6 weeks after primary immunization, and indirect antibody ELISA. Results Recombinant plasmids were cut into fragments of the corresponding size, confirmed by sequencing HSV-1gD sequence. Western blotting confirmed that it can be expressed in eukaryotic cells in vitro. After the mice were immunized, specific antibodies were produced with an antibody titer of 1: 2000. Conclusion HSV-1gD recombinant plasmid DNA may be used as a DNA vaccine against HSV-1 for the prevention and treatment of HSV-1 infection and related diseases. Key words herpes simplex virus keratitis DNA vaccine glycoprotein