本文選題：HSV + gC�。� 參考：《第四軍醫(yī)大學》2011年碩士論文

【摘要】：單純皰疹病毒(herpes simplex virus, HSV)是一種流行于全世界的皰疹性疾病病原體,根據病原性的差異分為HSV-1型和HSV-2型,HSV-1除引起常見的黏膜性皰疹外,還可引起皰疹性角膜炎和皰疹性腦炎,前者嚴重的可引起失明,后者則常伴有較重的神經系統(tǒng)后遺癥且可能引起死亡;HSV-2則主要通過性傳播引起常見的生殖器皰疹。近年的研究還顯示HSV感染可增強引起艾滋病(acquired immune deficiency syndrome, AIDS)的人類免疫缺陷病毒(human immunodeficiency virus, HIV)感染及傳播的幾率,因此,有效治療HSV感染對于預防HIV的傳播也具有重要的意義。目前對于HSV感染尚無有效的治療手段,臨床主要應用抑制病毒DNA復制類藥物進行治療,不能根除HSV的感染,且易產生耐藥性。用疫苗預防HSV的感染多處于臨床前研究階段,造成現(xiàn)有疫苗效果不佳的原因之一可能是病毒的免疫逃避分子對免疫系統(tǒng)的影響。HSV-1病毒糖蛋白C(gC-1)可與補體C3b結合,阻止補體系統(tǒng)的激活,糖蛋白E(gE)和糖蛋白I(gI)則能夠與抗體IgG的Fc結合,抑制補體的激活以及抗體依賴的細胞毒效應(ADCC)。近來有研究顯示用gC-1和gD-1聯(lián)合免疫對小鼠的保護作用要優(yōu)于gD-1單獨免疫,提示gC-1有可能用于增強疫苗的免疫效果。本研究在前期工作的基礎上,利用分子生物學技術,構建了HSV-1 gC的真核表達載體,在CHO-K1細胞和Sp2/0細胞中表達,并對其進行了初步鑒定。 1.構建了表達HSV-1 gC蛋白的真核表達載體PCI-neo-MARs-mCMV-gC1- IRES-DHFR-L22R 首先用MARs-mCMV替換PCI-neo載體的啟動子hCMV,陽性重組質粒命名為PCI-neo-MARs-mCMV;其次構建DHFR-L22R(即編碼第22位亮氨酸L的CTT用編碼精氨酸R的CGG替換)基因。將HSV-1 gC基因、IRES基因和DHFR-L22R基因先后克隆入經過改造的PCI-neo-MARs-mCMV載體,酶切鑒定獲得陽性重組質粒PCI-neo-MARs-mCMV-gC1-IRES-DHFR-L22R,大量制備重組質粒并測定DNA濃度與純度。 2.穩(wěn)定表達HSV-1 gC蛋白的CHO-K1細胞系與Sp2/0細胞系的建立與篩選 將大量制備的重組質粒PCI-neo-MARs-mCMV-gC1-IRES-DHFR-L22R按照Lipofectamine~(TM) 2000說明書轉染CHO-K1細胞與Sp2/0細胞,在G418和氨甲喋呤(MTX)雙篩選壓力下篩選穩(wěn)定轉染細胞系,Slot blot方法檢測表明細胞能穩(wěn)定表達目的蛋白HSV-1 gC。 3.HSV-1 gC蛋白的表達與鑒定 將篩選出的穩(wěn)定轉染細胞系培養(yǎng)并收集上清,超濾濃縮后用His-Ni瓊脂糖填料進行目的蛋白純化,純化后SDS-PAGE和Western blot分析表明,成功的表達了目的蛋白gC-1,且其具有良好的特異性結合活性,為進一步的實驗研究和臨床應用提供了基礎。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R373

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相關期刊論文 前2條

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本文編號：1777069