单纯疱疹病毒1型糖蛋白B截短基因疫苗的构建及初步免疫学研究

发布时间：2018-02-14 07:00

本文关键词： 单纯疱疹病毒1 型截短糖蛋白B 基因角膜炎基因疫苗　出处：《吉林大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:构建用于防治单纯疱疹病毒性角膜炎的核酸疫苗并进行初步免疫学研究,探讨HSV-1 截短糖蛋白B 基因作为基因疫苗的可能性。方法:利用PCR 技术从HSV-1 SM44 株基因组中扩增出HSV-gB 的截短基因片断gBt(1482bp),去掉了胞内区,跨膜区,信号肽中N 端39bp 和部分胞外区的基因序列,只包含部分信号肽序列和部分胞外区基因序列,将其定向插入真核表达质粒pcDNA3 载体中,构建出重组真核表达质粒pcDNA-gBt,并对其进行酶切分析、PCR 鉴定及测序鉴定;,用pcDNA-gBt 三次肌肉接种小鼠后,通过中和试验、ELISA 方法检测中和抗体滴度观察免疫效果,以单纯疱疹病毒攻击小鼠观察pcDNA-gBt 对小鼠的免疫保护作用。结果:双酶切重组质粒pcDNA-gBt,电泳可见两条带,分别为目的基因(1.5kb)和线性质粒pcDNA3(5.4kb);以重组质粒pcDNA-gBt 为模板进行PCR 扩增,在1.5kb 位置扩增出特异的产物;测序结果表明,克隆基因插入方向正确,与GeneBank 中登录的HSV-1F 株gB 基因序列比较,同源性达99.5%;实验小鼠产生了对HSV-1 特异性的抗体(抗体滴度:中和试验法1:61,ELISA 法1:406),以HSV-1 对小鼠进行病毒攻击(存活率80%)。结论:成功地构建了重组真核表达质粒单纯疱疹病毒糖蛋白B 截短基因疫苗,用其免疫小鼠可以诱导产生特异性免疫反应,并具有免疫保护作用;为进一步构建病毒糖蛋白联合疫苗并最终用于单疱病毒角膜炎的预防和治疗奠定理论基础。
[Abstract]:Aim: to construct a nucleic acid vaccine for the prevention and treatment of herpes simplex keratitis and to investigate the possibility of HSV-1 truncated glycoprotein B gene as gene vaccine. Methods: the truncated gene fragment of HSV-gB was amplified from the genome of HSV-1 SM44 strain by PCR technique, and the gene sequence of the N terminal and some extracellular region were removed, including the intracellular region, the transmembrane region, the N-terminal region and the partial extracellular region. Only some signal peptide sequences and some extracellular domain gene sequences were inserted into eukaryotic expression plasmid pcDNA3 vector. The recombinant eukaryotic expression plasmid pcDNA-gBtwas constructed, and the recombinant plasmid pcDNA-gBtwas identified by restriction endonuclease analysis and sequencing. After the mice were inoculated with pcDNA-gBt for three times, the titer of neutralizing antibody was detected by neutralization Elisa. The immune protective effect of pcDNA-gBt on mice was observed with herpes simplex virus (HSV) attack. Results: the recombinant plasmid pcDNA-gBt was digested with two bands, the target gene was 1.5 kb) and the linear plasmid pcDNA3 was 5.4 kb. The recombinant plasmid pcDNA-gBt was used as template to amplify the specific product at 1.5kb. The cloned gene was inserted in the correct direction and had 99.5 homology compared with the GB gene sequence of HSV-1F strain registered in GeneBank. The experimental mice produced specific antibody to HSV-1 (antibody titer: neutralization test 1: 61) Elisa method 1: 406, and HSV-1 was used to attack the mouse virus (survival rate 80%). Conclusion: the recombinant eukaryotic expression plasmid truncated gene vaccine of glycoprotein B of herpes simplex virus was successfully constructed. It lays a theoretical foundation for the further construction of virus glycoprotein combined vaccine and its application in the prevention and treatment of herpes simplex keratitis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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