单纯疱疹病毒1型糖蛋白C真核表达载体的构建及表达

发布时间：2018-04-20 08:42

本文选题：HSV + gC　；参考：《第四军医大学》2011年硕士论文

【摘要】：单纯疱疹病毒(herpes simplex virus, HSV)是一种流行于全世界的疱疹性疾病病原体,根据病原性的差异分为HSV-1型和HSV-2型,HSV-1除引起常见的黏膜性疱疹外,还可引起疱疹性角膜炎和疱疹性脑炎,前者严重的可引起失明,后者则常伴有较重的神经系统后遗症且可能引起死亡;HSV-2则主要通过性传播引起常见的生殖器疱疹。近年的研究还显示HSV感染可增强引起艾滋病(acquired immune deficiency syndrome, AIDS)的人类免疫缺陷病毒(human immunodeficiency virus, HIV)感染及传播的几率,因此,有效治疗HSV感染对于预防HIV的传播也具有重要的意义。目前对于HSV感染尚无有效的治疗手段,临床主要应用抑制病毒DNA复制类药物进行治疗,不能根除HSV的感染,且易产生耐药性。用疫苗预防HSV的感染多处于临床前研究阶段,造成现有疫苗效果不佳的原因之一可能是病毒的免疫逃避分子对免疫系统的影响。HSV-1病毒糖蛋白C(gC-1)可与补体C3b结合,阻止补体系统的激活,糖蛋白E(gE)和糖蛋白I(gI)则能够与抗体IgG的Fc结合,抑制补体的激活以及抗体依赖的细胞毒效应(ADCC)。近来有研究显示用gC-1和gD-1联合免疫对小鼠的保护作用要优于gD-1单独免疫,提示gC-1有可能用于增强疫苗的免疫效果。本研究在前期工作的基础上,利用分子生物学技术,构建了HSV-1 gC的真核表达载体,在CHO-K1细胞和Sp2/0细胞中表达,并对其进行了初步鉴定。 1.构建了表达HSV-1 gC蛋白的真核表达载体PCI-neo-MARs-mCMV-gC1- IRES-DHFR-L22R 首先用MARs-mCMV替换PCI-neo载体的启动子hCMV,阳性重组质粒命名为PCI-neo-MARs-mCMV;其次构建DHFR-L22R(即编码第22位亮氨酸L的CTT用编码精氨酸R的CGG替换)基因。将HSV-1 gC基因、IRES基因和DHFR-L22R基因先后克隆入经过改造的PCI-neo-MARs-mCMV载体,酶切鉴定获得阳性重组质粒PCI-neo-MARs-mCMV-gC1-IRES-DHFR-L22R,大量制备重组质粒并测定DNA浓度与纯度。 2.稳定表达HSV-1 gC蛋白的CHO-K1细胞系与Sp2/0细胞系的建立与筛选将大量制备的重组质粒PCI-neo-MARs-mCMV-gC1-IRES-DHFR-L22R按照Lipofectamine~(TM) 2000说明书转染CHO-K1细胞与Sp2/0细胞,在G418和氨甲喋呤(MTX)双筛选压力下筛选稳定转染细胞系,Slot blot方法检测表明细胞能稳定表达目的蛋白HSV-1 gC。 3.HSV-1 gC蛋白的表达与鉴定将筛选出的稳定转染细胞系培养并收集上清,超滤浓缩后用His-Ni琼脂糖填料进行目的蛋白纯化,纯化后SDS-PAGE和Western blot分析表明,成功的表达了目的蛋白gC-1,且其具有良好的特异性结合活性,为进一步的实验研究和临床应用提供了基础。
[Abstract]:Herpes simplex virus (HSV-1) is a worldwide herpes disease pathogen. It can be divided into HSV-1 type and HSV-2 type HSV-1 according to the pathogenicity of herpes simplex virus, which can cause herpes keratitis and herpes encephalitis in addition to common mucosal herpes. Severe blindness is associated with severe neurological sequelae and may cause death. HSV-2 mainly causes genital herpes through sexual transmission. Recent studies have also shown that HSV infection can enhance the infection and transmission of human immunodeficiency virus (HIV-1), which causes acquired immune deficiency syndrome (AIDSs). Therefore, the effective treatment of HSV infection is also of great significance in preventing the spread of HIV. At present, there is no effective treatment for HSV infection. The main clinical use of virus DNA replication drugs for treatment, can not eradicate the infection of HSV, and easy to produce drug resistance. The prevention of HSV infection with vaccine is mostly in the stage of preclinical study. One of the reasons for the poor effect of the existing vaccine may be that the immune escape molecule of the virus affects the immune system. HSV-1 glycoprotein CngC-1 can bind to complement C3b. The activation of complement system was blocked, and glycoprotein EguE and glycoprotein Igng I) could bind to FC of antibody IgG, inhibit the activation of complement and the cytotoxic effect of antibody dependent. Recent studies have shown that the protective effect of gC-1 and gD-1 combined immunization on mice is better than that of gD-1 alone, suggesting that gC-1 may be used to enhance the immune effect of the vaccine. On the basis of previous work, the eukaryotic expression vector of HSV-1 GC was constructed by molecular biology technique, and expressed in CHO-K1 cells and Sp2/0 cells. 1. The eukaryotic expression vector PCI-neo-MARs-mCMV-gC1- IRES-DHFR-L22R expressing HSV-1 GC protein was constructed. Firstly, the promoter of PCI-neo vector hCMV was replaced by MARs-mCMV, and the positive recombinant plasmid was named PCI-neo-MARs-mCMV. Secondly, the DHFR-L22R gene was constructed (that is, the CTT encoding leucine L at the 22nd position was replaced by CGG encoding arginine R). The IRES gene and DHFR-L22R gene of HSV-1 GC gene were cloned into the modified PCI-neo-MARs-mCMV vector successively. The positive recombinant plasmid PCI-neo-MARs-mCMV-gC1-IRES-DHFR-L22Rwas obtained by restriction endonuclease digestion. The recombinant plasmid was prepared and the concentration and purity of DNA were determined. 2. Establishment and screening of CHO-K1 and Sp2/0 Cell Lines stably expressing HSV-1 GC protein A large number of recombinant plasmid PCI-neo-MARs-mCMV-gC1-IRES-DHFR-L22R was transfected into CHO-K1 and Sp2/0 cells according to the instructions of Lipofectamineum 2000. The stable transfection cell lines were screened under double screening pressure of G418 and methotrexate. The results of blot blot analysis showed that the cells could express the target protein HSV-1 GC stably. Expression and Identification of 3.HSV-1 GC protein The stable transfected cell lines were cultured and the supernatants were collected. After ultrafiltration, the target protein was purified with His-Ni agarose filler. The results of SDS-PAGE and Western blot analysis showed that, The target protein gC-1 was successfully expressed and has good specific binding activity, which provides a basis for further experimental research and clinical application.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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