基于gE与IE63蛋白的水痘—带状疱疹病毒DNA疫苗的构建及其对小鼠免疫原性的研究

发布时间：2018-08-07 11:10

【摘要】：水痘(varicella)是高度传染性的儿童常见疾病,多发于2~6岁,冬春两季多发,其传染力强,传染源主要是患者,急性期患者的水痘内容物及呼吸道分泌物内均含有病毒。带状疱疹是一种常见于老年的急性感染性皮肤病,患者出现单侧性皮疹,按神经节段分布,伴有疼痛。本病春秋季节多见,发病率随年龄增大而呈显著上升。水痘与带状疱疹,虽然是两种不同的疾病,但却是由同一种病原体所引起,即水痘-带状疱疹病毒(varicella-zoster virus,VZV)。VZV在人体初次感染时引起水痘,恢复后病毒潜伏在体内,可长期潜伏于脊髓神经后根神经节的神经元内,在人体免疫力低下时复发引起带状疱疹,故被称为水痘-带状疱疹病毒。在中国,VZV的感染十分普遍,据统计在45岁以上人群中90%都是病毒的感染者或携带者。目前现有的疫苗分为两类:预防水痘的初次感染的疫苗被称为水痘疫苗,这类疫苗多是对儿童或者青年使用达到预防效果;预防病毒的复发的疫苗被称为带状疱疹疫苗,多对老年人使用。目前使用较普遍的这两类疫苗都是以减毒株VZV作为免疫原,针对不同的免疫目的,采用不同的配方和佐剂制成。这两类疫苗虽然都具有一定的保护效果,但都存在着病原体清除不彻底,细胞免疫应答低下等不足,为病毒的后续复发埋下隐患。gE蛋白(envelope glycoprotein E,gE)是VZV表面7种膜蛋白中的一种,在病毒感染以及传播过程中发挥重要的作用,研究显示,gE是病毒膜蛋白中最具有免疫原性的一个。但是目前基于gE蛋白构建的疫苗往往都只具有较强的体液免疫反应,而无法诱导较强的细胞免疫。前面说到VZV具有潜伏性,在人体免疫力低下的时候造成复发。因此对于VZV的预防,体液免疫与细胞免疫的双重保护很重要,其中细胞免疫更是尤为重要。为了使构建的疫苗在诱导体液免疫的同时也能产生相应的细胞免疫效果,我们在以gE为免疫原的基础上使用了DNA疫苗的形式,目的是为了增强疫苗诱导细胞免疫的能力,并引入了VZV的另一个蛋白——IE63蛋白(immediate early protein 63,IE63)。IE63蛋白是VZV早期复制所必须的一个功能性蛋白,病毒感染细胞后会大量表达IE63,同时IE63也是病毒在潜伏状态下唯一一个仍然高表达的蛋白。因此,针对IE63蛋白的疫苗有望引起较强的细胞免疫,并有望清除潜伏病毒。本文构建的DNA疫苗在pcDNA3.1质粒上插入外源片段gE和IE63序列,一共构建了3个重组质粒,3个重组质粒均能同时表达gE蛋白与IE63蛋白。以CpG寡聚脱氧核苷酸1826(CpG-ODN1826)为佐剂,对BALB/c小鼠进行腿部肌肉注射,经过3次免疫后,结果显示小鼠血液中产生了较强的gE特异性IgG抗体水平,通过对IgG抗体的亚型分析,以及对小鼠脾脏细胞干扰素γ分泌情况分析,结果显示我们构建的疫苗在诱导较强的体液免疫的同时也产生了相应的的T细胞免疫应答。通过本文的研究,我们验证了gE和IE63作为VZV重要抗原的地位。DNA疫苗形式很好的诱导了一种平衡性的细胞—体液免疫。对于水痘-带状疱疹的治疗与预防,提供了一种既注重防御病毒初次感染(抗体水平)又预防病毒复发(细胞免疫)的保护策略。同时,在疫苗设计中,对于体液—细胞平衡免疫的诱导,本文的做法也提供了一种可借鉴的思路。
[Abstract]:Chicken pox (varicella) is a common disease of highly contagious children. It often occurs in the age of 2~6. In the two quarter of winter and spring, the infectious force is strong. The infectious source is mainly the patient. The virus in the contents of chickenpox and the respiratory secretions in the acute period are all containing the virus. According to the ganglion segment distribution and pain, this disease is common in the spring and autumn season, and the incidence of the disease increases with age. The varicella and herpes zoster, although it is two different diseases, is caused by the same pathogen, that is, the varicella zoster virus (varicella-zoster virus, VZV).VZV causes chickenpox in the first infection of the human body. The retrovirus lurks in the body and can lurk in the neurons of the dorsal root ganglion of the spinal cord for a long time. It is known as varicella zoster virus in the low immunity of the human body. In China, the infection of VZV is very common. According to statistics, 90% of the people over 45 years old are the infected or carriers of the virus. The vaccine is divided into two categories: vaccines to prevent initial infection of chickenpox are called varicella vaccines. These vaccines are mostly used for children or young people to achieve preventive effects; the vaccine to prevent the recurrence of the virus is called herpes zoster vaccine and is used for older people. The two types of vaccines are currently used as immunogens with the strain VZV. According to different immunological purposes, different formulations and adjuvant are made. Although these two kinds of vaccines have certain protective effects, there are not thorough pathogen clearance, low cellular immune response and so on, and the hidden hidden danger.GE protein (envelope glycoprotein E, gE) is one of the 7 membrane proteins on the surface of VZV. Species, which play an important role in viral infection and transmission, have shown that gE is one of the most immunogenic in viral membrane proteins. However, the current vaccines based on gE protein often have strong humoral immune responses and can not induce strong cellular immunity. In the previous study, the VZV is latent and is immune to human immunity. Therefore, the double protection of humoral and cellular immunity is very important for the prevention of VZV, and the cellular immunity is particularly important. In order to induce the vaccine to induce humoral immunity, it can also produce the corresponding cellular immune effect. We use the DNA vaccine on the basis of gE as the immunogen. Form, the purpose is to enhance the ability of vaccine to induce cell immunity, and to introduce another protein of VZV - IE63 protein (immediate early protein 63, IE63).IE63 protein is a functional protein necessary for the early replication of VZV, and the virus infected cells will express IE63 in large amount, and IE63 is the only one of the virus in the latent state. It is still highly expressed protein. Therefore, the vaccine against IE63 protein is expected to cause strong cellular immunity, and is expected to clear the latent virus. The DNA vaccine constructed in this paper is inserted into the pcDNA3.1 plasmid by inserting exogenous fragment gE and IE63 sequence. Altogether 3 recombinant plasmids are constructed, and all 3 recombinant plasmids can simultaneously express gE protein and IE63 protein. CpG oligosaccharide oligoplasmids can be expressed simultaneously. Polydeoxynucleotides 1826 (CpG-ODN1826) was used as an adjuvant to injecting the leg muscles of BALB/c mice. After 3 immunizations, the results showed that a strong gE specific IgG antibody level was produced in the blood of the mice. The analysis of the subtype of IgG antibody and the analysis of the secretion of the interferon gamma in the spleen cells of the mice showed that the disease was constructed. The vaccine produced a corresponding T cell immune response while inducing strong humoral immunity. Through this study, we verified that the status of gE and IE63 as an important VZV antigen in the form of.DNA vaccine has induced a balanced cell humoral immunity. It provides a kind of treatment and prevention for varicella and herpes zoster. The protection strategy of defending the first infection of the virus (the level of the antibody) and preventing the recurrence of the virus (cell immunity) is also emphasized. Meanwhile, in the design of the vaccine, the practice of this paper also provides a useful way of thinking for the induction of the balance immunity of the body fluid to the cell.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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