本文選題：瘧原蟲 + 弓形蟲��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：人類許多疾病是由某種蛋白質(zhì)表達(dá)低于正常水平或者某種蛋白質(zhì)突變導(dǎo)致蛋白失活所引起的,針對這類疾病,目前的治療策略主要是通過補(bǔ)充適量相應(yīng)的蛋白質(zhì)或多肽達(dá)到緩解疾病癥狀[1][2]。比如人工合成胰島素治療糖尿病[3],能夠快速的緩解糖尿病癥狀及促進(jìn)糖尿病導(dǎo)致的并發(fā)癥快速的恢復(fù)。相比傳統(tǒng)化學(xué)藥物,生物制劑(蛋白及多肽類)具有針對性強(qiáng)、活性高、副作用小等優(yōu)點(diǎn),在目前的人類疾病治療中發(fā)揮了越來越重要作用。然而,目前應(yīng)用的臨床治療的蛋白質(zhì)及多肽類的生物制劑仍然較少,探究其主要原因有以下幾點(diǎn):(1)該類藥物屬于蛋白質(zhì)或多肽類分子,運(yùn)輸和儲存都需要低溫、低濕等嚴(yán)苛條件,一旦環(huán)境發(fā)生改變很容易導(dǎo)致藥物失活;(2)大部分蛋白質(zhì)及多肽在人體內(nèi)代謝速度快,半衰期短(一般是幾分鐘左右)[4],無法到達(dá)有效的血藥濃度。雖然目前通過改變其氨基酸序列、加側(cè)鏈、加用緩釋劑[5]等方法來延長其半衰期,但是仍然存在許多問題:改變其序列結(jié)構(gòu),不僅費(fèi)時費(fèi)力,并且極有可能導(dǎo)致其活性的降低甚至消失,也無法達(dá)到延長其半衰期的目的。半衰期問題的限制了很多蛋白質(zhì)及多肽應(yīng)用于臨床治療疾病。所以我們現(xiàn)在亟需要解決生物藥半衰期短的問題。(3)絕大部分生物制劑還只能通過靜脈或者皮下注射,半衰期短導(dǎo)致了患者經(jīng)常需要多次注射該類藥物,造成了依從性的下降。而這些問題都直接限制了該類藥物在臨床疾病治療中的使用。尋找一種能夠克服這些缺點(diǎn)的手段一直以來都是蛋白多肽類生物制劑研究的熱點(diǎn)。人體寄生蟲是與人類協(xié)同進(jìn)化而來的,為了適應(yīng)了人體內(nèi)的環(huán)境,寄生蟲已經(jīng)發(fā)展了多種免疫逃避和抑制機(jī)制,從而得以在宿主體內(nèi)存活,而且有些寄生蟲還可能處于長期感染狀態(tài)。隨著基因修飾技術(shù)的迅速發(fā)展,目前已經(jīng)可以對單細(xì)胞的寄生原蟲進(jìn)行成功的基因修飾,通過敲除某些原蟲發(fā)育階段的關(guān)鍵基因使其減毒,減緩其在人體內(nèi)的生長速度,甚至可以達(dá)到條件性控制原蟲在體內(nèi)的增殖。另外,原蟲在宿主體內(nèi)寄生的過程中,會主動分泌多種蛋白抗原及多肽類物質(zhì)進(jìn)入宿主血液中[6]。相對于原核的細(xì)菌,原蟲作為真核生物能夠正確的翻譯、折疊人源性蛋白質(zhì)及多肽;這些都提示我們原蟲具備與宿主長期共存、能夠準(zhǔn)確表達(dá)人源蛋白及毒性小等優(yōu)點(diǎn),對其進(jìn)行轉(zhuǎn)基因改造能夠使其成為宿主內(nèi)穩(wěn)定表達(dá)生物制劑的工具,從而克服現(xiàn)有生物制劑運(yùn)輸困難、半衰期短及多次給藥等缺點(diǎn),有望成為一種新的生物治療手段。本研究擬嘗試?yán)迷x中研究較多的瘧原蟲和弓形蟲為載體,通過CRISPR-Cas9基因編輯技術(shù)構(gòu)建穩(wěn)定表達(dá)鼠源瘦素(Leptin)蛋白的減毒約氏瘧原蟲,驗(yàn)證其能否表達(dá)鼠源Leptin蛋白,并探討其感染小鼠后,對小鼠體重的影響;同時,構(gòu)建穩(wěn)定表達(dá)人黑色素瘤抗原gp100蛋白的尿嘧啶缺陷型RH株弓形蟲,為今后探討其能否誘導(dǎo)小鼠產(chǎn)生gp100抗原特異性的抗腫瘤免疫反應(yīng)奠定基礎(chǔ)。一、感染瘦素蛋白轉(zhuǎn)基因約氏瘧原蟲顯著降低小鼠體重1.成功構(gòu)建含Leptin基因的瘧原蟲:通過在p YC框架質(zhì)粒(for plasmid for P.yoelii CRISPR/Cas9)中插入sg RNA序列,以及裝入帶Leptin基因的同源臂,然后將質(zhì)粒電轉(zhuǎn)進(jìn)入瘧原蟲體內(nèi),通過乙胺嘧啶抗性篩選出帶有p YC重組質(zhì)粒的陽性瘧原蟲,再通過限制性稀釋法成功篩選出轉(zhuǎn)基因成功的單克隆的瘧原蟲。2.轉(zhuǎn)基因瘧原蟲能表達(dá)Leptin:通過提取瘧原蟲的c DNA,然后通過擴(kuò)增得到MIF-Leptin基因,送上海英俊公司測序,測序結(jié)果顯示MIF與Leptin基因融合轉(zhuǎn)錄;然后用mouse Leptin抗體對瘧原蟲進(jìn)行間接免疫熒光染色,結(jié)果發(fā)現(xiàn)轉(zhuǎn)基因瘧原蟲能表達(dá)瘦素蛋白。3.感染轉(zhuǎn)基因瘧原蟲顯著降低小鼠體重:分別通過腹腔注射轉(zhuǎn)基因瘧原蟲、野生型瘧原蟲感染C57小鼠,對照組注射等量PBS溶液,隨后隔天檢測其原蟲血癥及體重并記錄;最后統(tǒng)計學(xué)分析原蟲血癥與體重的關(guān)系,結(jié)果顯示原蟲血癥在一定范圍內(nèi),轉(zhuǎn)基因瘧原蟲能夠降低C57/BL6小鼠體重,而野生型瘧原蟲不能。二、成功構(gòu)建表達(dá)黑色素瘤抗原gp100的轉(zhuǎn)基因減毒弓形蟲1.成功構(gòu)建弓形蟲CRISPR-Cas9質(zhì)粒和同源臂質(zhì)粒:通過高保真酶對框架質(zhì)粒(p SAG1-Cas9-U6-sg UPRT)進(jìn)行擴(kuò)增,然后環(huán)化,將sg RNA序列裝入框架質(zhì)粒中;通過重疊PCR的方法構(gòu)建同源臂并插入重組質(zhì)粒中。2.成功篩選和克隆表達(dá)gp100的轉(zhuǎn)基因減毒弓形蟲:將上述構(gòu)建好的質(zhì)粒電轉(zhuǎn)進(jìn)入弓形蟲體內(nèi),然后通過乙胺嘧啶抗性篩選出電轉(zhuǎn)成功的弓形蟲。本研究主要嘗試以人體寄生蟲中的瘧原蟲弱毒株和減毒弓形蟲作為載體表達(dá)和分泌宿主蛋白質(zhì)。通過了CRISPR/Cas9基因編輯技術(shù)將小鼠瘦素基因插入瘧原蟲基因組中,經(jīng)檢測能夠轉(zhuǎn)基因弱毒瘧原蟲能夠轉(zhuǎn)錄和表達(dá)瘦素蛋白,其感染小鼠后并能夠降低宿主小鼠的體重;另外,成功構(gòu)建能將gp100基因轉(zhuǎn)入弓形蟲基因的CRISPR/Cas9質(zhì)粒,然后將gp100基因插入到弓形蟲基因組中,從而構(gòu)建能表達(dá)gp100的減毒弓形蟲,為后續(xù)探討其能否誘導(dǎo)抗腫瘤特異性免疫奠定基礎(chǔ)。本研究中,通過基因組編輯技術(shù)構(gòu)建了減毒原蟲作為載體表達(dá)宿主蛋白,為探索新的人體內(nèi)重組蛋白表達(dá)工具提供新的思路和理論依據(jù)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R3411

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