疟原虫种间鉴定及恶性疟原虫子孢子表面蛋白质功能分析

发布时间：2018-06-20 22:06

本文选题：疟原虫 + 种间鉴定　；参考：《吉林大学》2011年博士论文

【摘要】：疟原虫是一种肉眼看不到的单细胞生物,属于真球虫目,疟原虫科,疟原虫属,是疟疾的病原体。目前世界上近23亿人处于感染疟疾的危险之中。每年约有2.5亿感染,死亡人数近100万。疟疾在非洲流行一直非常严重,每年由疟疾导致的儿童死亡率占儿童总死亡率的20%(2009年3月WHO)。虽然科学家对疟疾疫苗的研制一直没有中断,但还没有研制出一种值得信赖的疟疾疫苗。同时由于疟原虫耐药性和按蚊抗药性的增加,目前已经没有十分有效的药物应对疟疾的流行。基于以上问题本课题进行了两方面的工作。第一部分疟原虫的种间鉴定常见的感染人的疟原虫有4种,它们分别是间日疟原虫(P. vivax)、恶性疟原虫(P. falciparum)、三日疟原虫(P. malariae)和卵形疟原虫(P. ovalee)。2004年,又发现一种疟原虫可以感染人类,即诺氏疟原虫(P. knowlesi)。它原本是一种感染长尾猕猴的疟原虫,但最近在马来西亚婆罗洲、泰国、缅甸和菲律宾等地已经发现了诺氏疟原虫感染人的病例并引起死亡。因此,诺氏疟原虫被认为是感染人的第五种疟原虫,同时诺氏疟也成为了一种人兽共患病。 2008年,云南省寄生虫病防治所送检了146份疟原虫病人的血液样品。这些样品均来自于与中国云南边境相接壤的缅甸境内。本试验应用一种巢式PCR检测方法,以核糖体小亚基18S RNA基因(18S SSU rRNA,18S)为参考序列,分别检测寄生于人体的五种疟原虫P. vivax, P.falciparum, P. malariae, P. ovalee和P. knowlesi。在检测过程中发现用于检测诺氏疟原虫的引物pmk8和pmkr9扩增的条带不是十分特异,影响了诺氏疟原虫检测结果的准确性。因此本试验应用另一个子孢子表面重要蛋白-环子孢子蛋白(circumsporozoite protein, CSP)为参考基因设计引物进行诺氏疟原虫的验证检测,以辅助核糖体小亚基18S RNA基因的检测。结果在该地区首次检测到诺氏疟原虫感染人的病例,研究结果对我国疟疾防控措施的制定具有重要意义。鉴于在该地区首次鉴定到诺氏疟原虫感染人的病例,我们在当地采集了45份猴的血样,以期对该地区猴的诺氏疟原虫感染情况进行调查,结果在猴的血液样品中并未检测到任何一种疟原虫。第二部分恶性疟原虫子孢子表面蛋白功能分析恶性疟原虫是到目前为止最致命的一种疟原虫,绝大多数疟疾死亡病例是由恶性疟原虫引起的。恶性疟疾由雌性按蚊叮咬释放疟原虫子孢子传播引起,疟原虫子孢子一旦进入血液循环,迅速侵入肝细胞,然后侵入红细胞,导致疟疾的发作。子孢子侵入肝细胞是疟疾感染的关键,阻断子孢子的侵入,即可防止疟疾感染。子孢子表面蛋白CSP和血凝素相关黏附蛋白(thrombospondin-related anonymous protein, TRAP)是子孢子表面表达量最多的蛋白,在子孢子入侵肝细胞过程中具有重要的作用。研究CSP和TRAP的免疫原性对子孢子疫苗的研制具有重要意义。子孢子入侵肝细胞的机制尚未完全弄清楚,但入侵的关键点是子孢子表面的配体蛋白与肝细胞表面的受体蛋白发生特异性反应。CSP和TRAP被认为是子孢子入侵肝细胞的配体蛋白,理由是CSP和TRAP均位于子孢子表面,且它们的抗体能抑制子孢子入侵肝细胞。CD81分子是表达于肝细胞表面的一种膜蛋白,CD81缺陷型小鼠不能感染疟原虫。所以CD81可能是肝细胞表面的受体蛋白。因此本试验围绕着恶性疟原虫子孢子表面蛋白的免疫原性和子孢子入侵肝细胞过程中受体和配体的相互作用展开。根据恶性疟原虫子孢子表面蛋白质CSP和TRAP的结构和功能设计特异性的引物,将CSP分成3段,分别为C1,C2和C3：将TRAP分成2段,分别为T1和T2。分别构建了原核表达载体pGEX-4T-1-C1, pGEX-4T-1-C2 pGEX-4T-1-C3, pGEX-4T-1-T1和pGEX-4T-1-T2。将上述质粒分别转化入BL21-CodonPlus(DE3)-RIPL中。同时根据CD81大胞外区(CD81-LEL)的序列设计特异性引物,构建了原核表达载体pET22b-CD81-LEL并转化入Rosetta(DE3)中。对鉴定正确的上述重组菌分别应用IPTG进行诱导表达,经SDS-PAGE和Western blot进行鉴定,优化表达条件。再分别应用Glutathione SepharoseTM 4B和His GraviTrap进行纯化,经过透析除盐,BCA法进行浓度的测定,得到纯度和浓度均较高的重组蛋白质为研究恶性疟原虫子孢子表面蛋白免疫原性和子孢子入侵入肝细胞的机制提供物质储备。在免疫原性分析中采用弗氏佐剂分别与子孢子表面重组蛋白质C1, C2 C3, T1和T2混合乳化免疫Wistar大鼠,通过应用间接ELISA方法比较不同重组蛋白组中免疫血清的抗体动态变化、抗体效价、抗体IgG亚型来确定其免疫原性的强弱。发现这些子孢子表面蛋白在大鼠体内均能产生特异性的抗体,在3免后抗体效价达到高点,并能持续一定的时间。所有的子孢子表面蛋白在大鼠内产生IgGl/IgG2a的比值均小于1,但接近1。表明以Th1和Th2混合反应为主。为恶性疟原虫子孢子表面重组蛋白质疫苗的研究奠定了基础。在入侵机制研究中将分段表达纯化的恶性疟原虫子孢子表面蛋白C1, C2 C3, T1, T2和GST蛋白与人类肝细胞进行黏附试验,证实了CSP和TRAP的各段蛋白具有与HepG2细胞黏附的特性,其中C3和T2与HepG2细胞的结合能力相对较强。将这些蛋白分别与纯化的肝细胞表面蛋白CD81-LEL进行结合试验,通过GST-pulldown和His-pulldown证明了重组表达的子孢子表面蛋白C2与CD81的大胞外区具有结合作用。从而在蛋白水平上确定了CD81的大胞外区与C2蛋白具有结合能力。该试验为子孢子入侵肝细胞机制的研究提供了新的线索。
[Abstract]:The malaria parasite is an invisible single celled organism, belonging to the coccidiorders, Plasmodium, Plasmodium, and malaria pathogens. Nearly 2 billion 300 million people in the world are at risk of malaria infection. There are about 250 million infections and nearly 1 million deaths each year. Malaria has been very serious in Africa and died of malaria caused by malaria every year. The death rate accounts for 20% of the total child mortality rate (WHO March 2009). Although scientists have not disrupted the development of the malaria vaccine, a reliable malaria vaccine has not been developed. At the same time, there has been no very effective drug response to malaria due to the resistance of malaria parasites and the increase of Anopheles insecticide resistance. The subject has been carried out in two aspects.
Interspecific identification of the first part of the Plasmodium
There are 4 common parasites infected with Plasmodium vivax (P. vivax), Plasmodium falciparum (P. falciparum), three day Plasmodium (P. malariae) and Plasmodium oval (P. ovalee).2004 years, and a kind of Plasmodium can infect humans, namely, Plasmodium Nobel (P. knowlesi). It was originally a kind of parasite infected with long tailed rhesus macaques. But recently, in Malaysia, Borneo, Thailand, Burma and Philippines, the malaria parasite has been found to have been infected and killed. Therefore, Plasmodium norroshi is considered to be infected with fifth kinds of Plasmodium, and the malaria parasite has also become a zoonosis.
In 2008, the parasitic disease control Institute of Yunnan province examined 146 blood samples from the malaria parasite patients. All of these samples were from the Burma border with China's Yunnan border. The test used a nested PCR detection method, using the ribosome small subunit 18S RNA gene (18S SSU rRNA, 18S) as the reference sequence to detect five parasites in the human body. The detection of Plasmodium P. vivax, P.falciparum, P. malariae, P. ovalee and P. knowlesi. showed that the primer pmk8 and pmkr9 amplified bands used for the detection of Plasmodium Nobel were not very specific, affecting the accuracy of the detection results of Plasmodium Nobel. Therefore, this test should be used as an important protein of the surface of another sporozoite - circular spore. Circumsporozoite protein (CSP) was used as a reference gene to design primers for detection of Plasmodium Nobel, to assist the detection of the small subunit 18S RNA gene of ribosome. The results were the first to detect the cases of Plasmodium nosuriaris infection in this area. The results were of great significance to the formulation of malaria control measures in China.
In view of the first identification of the cases of Plasmodium Nobel infection in the area, we collected 45 samples of the blood samples from the local monkeys to investigate the infection of the malaria parasite in the monkeys, and the results were not detected in any of the malaria parasites in the monkey's blood samples.
The second part is functional analysis of spore surface proteins of Plasmodium falciparum.
Plasmodium falciparum is the most fatal Plasmodium to date, and most of the deaths of malaria are caused by Plasmodium falciparum. Malarial malaria is caused by the transmission of sporozoite sporozoites from the bites of Anopheles females. Once the sporozoites of the parasite enter the blood circulation, they quickly invade the liver cells and invade red blood cells, causing malaria hair. Subspore invasion of liver cells is the key to malaria infection. Blocking the intruded spores can prevent malaria infection. Subspore surface protein CSP and hemagglutinin associated adhesion protein (thrombospondin-related anonymous protein, TRAP) are the most expressed egg white on the surface of subspore, which are important in the process of subspore invasion of liver cells. The immunogenicity of CSP and TRAP is of great importance to the development of sporozoites.
The mechanism of the subspore invasion of the liver cells is not completely clear, but the key point of the invasion is that the ligand protein of the subspore surface is specific to the receptor protein on the liver cell surface.CSP and TRAP is considered to be the ligand protein of the subspore invasion of the liver cells. The reason is that CSP and TRAP are located on the surface of the sporozoites, and their antibodies can be suppressed. Sporozoite invading liver cells.CD81 molecule is a membrane protein expressed on the surface of liver cells, CD81 deficient mice infected with the parasite cannot. So CD81 may be a receptor protein on the surface of hepatocytes.
Therefore, the experiment focused on the immunogenicity of spore surface proteins of Plasmodium falciparum and the interaction of receptors and ligands in the process of sporozoites invading hepatocytes.
Specific primers were designed based on the structure and function of the surface protein CSP and TRAP of sporozoites of Plasmodium falciparum. CSP was divided into 3 segments, which were C1, C2 and C3, respectively. TRAP was divided into 2 segments, and T1 and T2. respectively constructed the prokaryotic expression vector pGEX-4T-1-C1, pGEX-4T-1-C2 pGEX-4T-1-C3, respectively. In BL21-CodonPlus (DE3) -RIPL, specific primers were designed based on the sequence of CD81 large extracellular domain (CD81-LEL), and the prokaryotic expression vector pET22b-CD81-LEL was constructed and transformed into Rosetta (DE3). IPTG was used to induce the identification of the correct recombinant bacteria. The expression was identified by SDS-PAGE and Western blot, and the expression conditions were optimized. Glutathione SepharoseTM 4B and His GraviTrap were used to purify each other, and the concentration was determined by dialysate desalination and BCA method. The recombinant protein with high purity and high concentration was obtained to provide material reserve for the mechanism of the immunogenicity of the surface protein of Plasmodium sporozoites and the mechanism of subspore invasion into the liver.
In the immunogenicity analysis, Freund's adjuvant was used to immunization Wistar rats with recombinant protein C1, C2 C3, T1 and T2, respectively. By using indirect ELISA method, the antibody dynamic changes, antibody titer and antibody IgG subtype were used to determine the immunogenicity of the immune sera in different recombinant protein groups. The surface protein of subspore surface can produce specific antibodies in rats. After 3 exempts, the titer of the antibody reaches a high point and can continue for a certain time. The ratio of all subspore surface proteins to IgGl/IgG2a in rats is less than 1, but close to 1. indicates that the mixture of Th1 and Th2 is the main surface recombinant egg of Plasmodium falciparum. The research of the white matter vaccine lays the foundation.
The protein C1, C2 C3, T1, T2 and GST protein were adhered to the human liver cells by subsection expression of the purified Plasmodium spore surface protein, which confirmed that the proteins of CSP and TRAP were adhered to HepG2 cells, and the binding ability of C3 and T2 to HepG2 fine cells was relatively strong. The purified hepatocyte surface protein CD81-LEL binding test, through GST-pulldown and His-pulldown, demonstrated that the recombinant expression of the subspore surface protein C2 was binding to the large extracellular domain of CD81, thus determining the binding ability of the large extracellular domain of CD81 to the C2 protein at the protein level. The test was a subspore invasion of the liver cell machine. The study of the system provides new clues.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R382.31

【参考文献】