稳定表达外源蛋白的转基因遗传减毒原虫治疗疾病的初步实验研究

发布时间：2018-06-21 19:21

本文选题：疟原虫 + 弓形虫　；参考：《第三军医大学》2017年硕士论文

【摘要】：人类许多疾病是由某种蛋白质表达低于正常水平或者某种蛋白质突变导致蛋白失活所引起的,针对这类疾病,目前的治疗策略主要是通过补充适量相应的蛋白质或多肽达到缓解疾病症状[1][2]。比如人工合成胰岛素治疗糖尿病[3],能够快速的缓解糖尿病症状及促进糖尿病导致的并发症快速的恢复。相比传统化学药物,生物制剂(蛋白及多肽类)具有针对性强、活性高、副作用小等优点,在目前的人类疾病治疗中发挥了越来越重要作用。然而,目前应用的临床治疗的蛋白质及多肽类的生物制剂仍然较少,探究其主要原因有以下几点:(1)该类药物属于蛋白质或多肽类分子,运输和储存都需要低温、低湿等严苛条件,一旦环境发生改变很容易导致药物失活;(2)大部分蛋白质及多肽在人体内代谢速度快,半衰期短(一般是几分钟左右)[4],无法到达有效的血药浓度。虽然目前通过改变其氨基酸序列、加侧链、加用缓释剂[5]等方法来延长其半衰期,但是仍然存在许多问题:改变其序列结构,不仅费时费力,并且极有可能导致其活性的降低甚至消失,也无法达到延长其半衰期的目的。半衰期问题的限制了很多蛋白质及多肽应用于临床治疗疾病。所以我们现在亟需要解决生物药半衰期短的问题。(3)绝大部分生物制剂还只能通过静脉或者皮下注射,半衰期短导致了患者经常需要多次注射该类药物,造成了依从性的下降。而这些问题都直接限制了该类药物在临床疾病治疗中的使用。寻找一种能够克服这些缺点的手段一直以来都是蛋白多肽类生物制剂研究的热点。人体寄生虫是与人类协同进化而来的,为了适应了人体内的环境,寄生虫已经发展了多种免疫逃避和抑制机制,从而得以在宿主体内存活,而且有些寄生虫还可能处于长期感染状态。随着基因修饰技术的迅速发展,目前已经可以对单细胞的寄生原虫进行成功的基因修饰,通过敲除某些原虫发育阶段的关键基因使其减毒,减缓其在人体内的生长速度,甚至可以达到条件性控制原虫在体内的增殖。另外,原虫在宿主体内寄生的过程中,会主动分泌多种蛋白抗原及多肽类物质进入宿主血液中[6]。相对于原核的细菌,原虫作为真核生物能够正确的翻译、折叠人源性蛋白质及多肽;这些都提示我们原虫具备与宿主长期共存、能够准确表达人源蛋白及毒性小等优点,对其进行转基因改造能够使其成为宿主内稳定表达生物制剂的工具,从而克服现有生物制剂运输困难、半衰期短及多次给药等缺点,有望成为一种新的生物治疗手段。本研究拟尝试利用原虫中研究较多的疟原虫和弓形虫为载体,通过CRISPR-Cas9基因编辑技术构建稳定表达鼠源瘦素(Leptin)蛋白的减毒约氏疟原虫,验证其能否表达鼠源Leptin蛋白,并探讨其感染小鼠后,对小鼠体重的影响;同时,构建稳定表达人黑色素瘤抗原gp100蛋白的尿嘧啶缺陷型RH株弓形虫,为今后探讨其能否诱导小鼠产生gp100抗原特异性的抗肿瘤免疫反应奠定基础。一、感染瘦素蛋白转基因约氏疟原虫显著降低小鼠体重1.成功构建含Leptin基因的疟原虫:通过在p YC框架质粒(for plasmid for P.yoelii CRISPR/Cas9)中插入sg RNA序列,以及装入带Leptin基因的同源臂,然后将质粒电转进入疟原虫体内,通过乙胺嘧啶抗性筛选出带有p YC重组质粒的阳性疟原虫,再通过限制性稀释法成功筛选出转基因成功的单克隆的疟原虫。2.转基因疟原虫能表达Leptin:通过提取疟原虫的c DNA,然后通过扩增得到MIF-Leptin基因,送上海英俊公司测序,测序结果显示MIF与Leptin基因融合转录;然后用mouse Leptin抗体对疟原虫进行间接免疫荧光染色,结果发现转基因疟原虫能表达瘦素蛋白。3.感染转基因疟原虫显著降低小鼠体重:分别通过腹腔注射转基因疟原虫、野生型疟原虫感染C57小鼠,对照组注射等量PBS溶液,随后隔天检测其原虫血症及体重并记录;最后统计学分析原虫血症与体重的关系,结果显示原虫血症在一定范围内,转基因疟原虫能够降低C57/BL6小鼠体重,而野生型疟原虫不能。二、成功构建表达黑色素瘤抗原gp100的转基因减毒弓形虫1.成功构建弓形虫CRISPR-Cas9质粒和同源臂质粒:通过高保真酶对框架质粒(p SAG1-Cas9-U6-sg UPRT)进行扩增,然后环化,将sg RNA序列装入框架质粒中;通过重叠PCR的方法构建同源臂并插入重组质粒中。2.成功筛选和克隆表达gp100的转基因减毒弓形虫:将上述构建好的质粒电转进入弓形虫体内,然后通过乙胺嘧啶抗性筛选出电转成功的弓形虫。本研究主要尝试以人体寄生虫中的疟原虫弱毒株和减毒弓形虫作为载体表达和分泌宿主蛋白质。通过了CRISPR/Cas9基因编辑技术将小鼠瘦素基因插入疟原虫基因组中,经检测能够转基因弱毒疟原虫能够转录和表达瘦素蛋白,其感染小鼠后并能够降低宿主小鼠的体重;另外,成功构建能将gp100基因转入弓形虫基因的CRISPR/Cas9质粒,然后将gp100基因插入到弓形虫基因组中,从而构建能表达gp100的减毒弓形虫,为后续探讨其能否诱导抗肿瘤特异性免疫奠定基础。本研究中,通过基因组编辑技术构建了减毒原虫作为载体表达宿主蛋白,为探索新的人体内重组蛋白表达工具提供新的思路和理论依据。
[Abstract]:Many human diseases are caused by a certain protein expression below normal level or a protein mutation causing protein inactivation. For these diseases, the current treatment strategy is to alleviate the symptoms of the disease by supplementing appropriate amounts of appropriate protein or polypeptide to alleviate the symptoms of [1][2]., such as artificial synthetic insulin for diabetic [3]. Rapid relief of diabetic symptoms and rapid recovery of complications caused by diabetes. Compared with traditional chemical drugs, biological agents (proteins and peptides) have the advantages of strong targeted, high activity, and small side effects, which are playing an increasingly important role in the current treatment of human disease. However, the clinical therapeutic proteins are currently used. The main reasons for the quality and polypeptide are still few, and the main reasons are as follows: (1) the drugs belong to protein or polypeptide molecules, and transport and storage require low temperature, low humidity and other harsh conditions. Once the environment changes, the drug is easily inactivated; (2) most of the proteins and peptides are rapidly metabolized in the human body and half decline. [4], which is short (usually a few minutes or so), can not reach the effective blood concentration. Although the amino acid sequence, the side chain and the slow release agent [5] are used to prolong the half-life, there are still many problems: changing its sequence structure is not only time-consuming and laborious, but also may lead to the decrease or even disappearance of its activity. The half-life problem has limited the application of many proteins and peptides to the clinical treatment of diseases. So we now need to solve the problem of short half-life. (3) most biologics can only be injected through the veins or skin, and the short half-life has caused the patient to need many times. The injection of such drugs has caused a decline in compliance. These problems have directly restricted the use of such drugs in the treatment of clinical diseases. Finding a means to overcome these shortcomings has always been a hot spot in the research of protein and polypeptide biologics. Human parasites are coevolved with human beings to adapt to people. In the body's environment, parasites have developed a variety of immune escape and inhibition mechanisms to survive in the host, and some parasites may be in a long-term infection state. With the rapid development of gene modification technology, a successful gene modification of single cell parasites has been made, by knocking out some raw materials. The key genes in the development stage of the insect make it detoxification, slow down their growth rate in the human body and even achieve the conditional control of the proliferation of protozoa in the body. In addition, in the host parasitic process, the protozoa will actively secrete a variety of protein antigens and polypeptide substances into the host blood of [6]. relative to the prokaryotic bacteria, protozoa as a parasite. Eukaryotes can correctly translate, fold human proteins and peptides, all of which suggest that our protozoa can coexist with the host for a long time, and can accurately express human protein and small toxicity. It can be transformed into a tool for the stable expression of biological agents in the host, thus overcoming the transportation of the existing biological agents. Difficulties, short half-life and many drug delivery are expected to be a new means of biological treatment. This study intends to try to use the Plasmodium and Toxoplasma gondii as carriers in the protozoa and construct the Plasmodium Plasmodium Plasmodium by CRISPR-Cas9 gene editing technique to express Leptin protein, which can express mouse source Le The effect of PTIN protein on the weight of mice after infection in mice, and the construction of an uracil deficient RH strain, which stably expressed human melanoma antigen gp100 protein, was the basis for the future study on whether it could induce the antitumor immune response of the mice to produce gp100 antigen specificity. The insect significantly reduced the weight of the mice by 1.. The Plasmodium containing the Leptin gene was successfully constructed: the SG RNA sequence was inserted in the P YC framework plasmid (for plasmid for P.yoelii CRISPR/Cas9) and the homologous arm loaded with Leptin gene, then the plasmid was transferred into the Plasmodium, and the recombinant plasmid with the recombinant plasmid was screened through the pyrimidine resistance. Plasmodium Plasmodium, which successfully screened the Plasmodium Plasmodium.2. transgenic by the restrictive dilution method, could express the Leptin: by extracting the C DNA of the Plasmodium, and then the MIF-Leptin gene was amplified by the amplification of the MIF-Leptin gene. The sequencing results showed that the MIF and the Leptin gene were transcribed, and then mouse Lepti was used. Indirect immunofluorescence staining of N antibody showed that transgenic Plasmodium Plasmodium could significantly reduce the weight of transgenic Plasmodium infected by transgenic Plasmodium.3. by intraperitoneal injection of transgenic Plasmodium, C57 mice infected with wild type Plasmodium, and PBS solution of the control group, and then detected the parasiemia after the other day. The relationship between the weight of protozoa and protozoa was statistically analyzed. The results showed that the transgenic Plasmodium could reduce the weight of C57/BL6 mice in a certain range, while the wild type Plasmodium could not. Two, a successful construction of the Antitoxoplasma Toxoplasma 1. successfully constructed the Toxoplasma gondii 1. to construct the Toxoplasma gondii CRISPR-Cas9 Plasmid of grain and homologous arm: P SAG1-Cas9-U6-sg UPRT was amplified by high fidelity enzyme, and then cyclized, SG RNA sequence was loaded into the frame plasmid. By constructing the homologous arm and inserting the recombinant plasmid into the recombinant plasmid by overlapping PCR,.2. successfully screened and cloned the genetically modified Toxoplasma gondii expressing gp100: the above constructed plasmid electricity. The main attempt was to express and secrete the host protein by using the Plasmodium Plasmodium and the Toxoplasma gondii as carriers in human parasites. The CRISPR/Cas9 gene editing technique was used to insert the mouse leptin gene into the Plasmodium genome. After detection, transgenic Plasmodium Plasmodium can transcribe and express leptin protein, which can infect mice and reduce the body weight of the host mice. In addition, the gp100 gene can be successfully constructed to transfer the CRISPR/Cas9 plasmid into the Toxoplasma gondii gene, and then the gp100 gene is inserted into the genome of Toxoplasma gondii to construct a reduced toxoplasmosis capable of expressing gp100. In this study, we constructed the antivirus protozoa as the carrier to express the host protein by genome editing technique, and provided a new idea and theoretical basis for exploring the new recombinant protein expression tool in human body.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3411

【参考文献】