本文選題：瘧原蟲 + 種間鑒定�。� 參考：《吉林大學(xué)》2011年博士論文

【摘要】：瘧原蟲是一種肉眼看不到的單細(xì)胞生物,屬于真球蟲目,瘧原蟲科,瘧原蟲屬,是瘧疾的病原體。目前世界上近23億人處于感染瘧疾的危險(xiǎn)之中。每年約有2.5億感染,死亡人數(shù)近100萬。瘧疾在非洲流行一直非常嚴(yán)重,每年由瘧疾導(dǎo)致的兒童死亡率占兒童總死亡率的20%(2009年3月WHO)。雖然科學(xué)家對瘧疾疫苗的研制一直沒有中斷,但還沒有研制出一種值得信賴的瘧疾疫苗。同時由于瘧原蟲耐藥性和按蚊抗藥性的增加,目前已經(jīng)沒有十分有效的藥物應(yīng)對瘧疾的流行�；谝陨蠁栴}本課題進(jìn)行了兩方面的工作。 第一部分瘧原蟲的種間鑒定 常見的感染人的瘧原蟲有4種,它們分別是間日瘧原蟲(P. vivax)、惡性瘧原蟲(P. falciparum)、三日瘧原蟲(P. malariae)和卵形瘧原蟲(P. ovalee)。2004年,又發(fā)現(xiàn)一種瘧原蟲可以感染人類,即諾氏瘧原蟲(P. knowlesi)。它原本是一種感染長尾獼猴的瘧原蟲,但最近在馬來西亞婆羅洲、泰國、緬甸和菲律賓等地已經(jīng)發(fā)現(xiàn)了諾氏瘧原蟲感染人的病例并引起死亡。因此,諾氏瘧原蟲被認(rèn)為是感染人的第五種瘧原蟲,同時諾氏瘧也成為了一種人獸共患病。 2008年,云南省寄生蟲病防治所送檢了146份瘧原蟲病人的血液樣品。這些樣品均來自于與中國云南邊境相接壤的緬甸境內(nèi)。本試驗(yàn)應(yīng)用一種巢式PCR檢測方法,以核糖體小亞基18S RNA基因(18S SSU rRNA,18S)為參考序列,分別檢測寄生于人體的五種瘧原蟲P. vivax, P.falciparum, P. malariae, P. ovalee和P. knowlesi。在檢測過程中發(fā)現(xiàn)用于檢測諾氏瘧原蟲的引物pmk8和pmkr9擴(kuò)增的條帶不是十分特異,影響了諾氏瘧原蟲檢測結(jié)果的準(zhǔn)確性。因此本試驗(yàn)應(yīng)用另一個子孢子表面重要蛋白-環(huán)子孢子蛋白(circumsporozoite protein, CSP)為參考基因設(shè)計(jì)引物進(jìn)行諾氏瘧原蟲的驗(yàn)證檢測,以輔助核糖體小亞基18S RNA基因的檢測。結(jié)果在該地區(qū)首次檢測到諾氏瘧原蟲感染人的病例,研究結(jié)果對我國瘧疾防控措施的制定具有重要意義。 鑒于在該地區(qū)首次鑒定到諾氏瘧原蟲感染人的病例,我們在當(dāng)?shù)夭杉?5份猴的血樣,以期對該地區(qū)猴的諾氏瘧原蟲感染情況進(jìn)行調(diào)查,結(jié)果在猴的血液樣品中并未檢測到任何一種瘧原蟲。 第二部分惡性瘧原蟲子孢子表面蛋白功能分析 惡性瘧原蟲是到目前為止最致命的一種瘧原蟲,絕大多數(shù)瘧疾死亡病例是由惡性瘧原蟲引起的。惡性瘧疾由雌性按蚊叮咬釋放瘧原蟲子孢子傳播引起,瘧原蟲子孢子一旦進(jìn)入血液循環(huán),迅速侵入肝細(xì)胞,然后侵入紅細(xì)胞,導(dǎo)致瘧疾的發(fā)作。子孢子侵入肝細(xì)胞是瘧疾感染的關(guān)鍵,阻斷子孢子的侵入,即可防止瘧疾感染。子孢子表面蛋白CSP和血凝素相關(guān)黏附蛋白(thrombospondin-related anonymous protein, TRAP)是子孢子表面表達(dá)量最多的蛋白,在子孢子入侵肝細(xì)胞過程中具有重要的作用。研究CSP和TRAP的免疫原性對子孢子疫苗的研制具有重要意義。 子孢子入侵肝細(xì)胞的機(jī)制尚未完全弄清楚,但入侵的關(guān)鍵點(diǎn)是子孢子表面的配體蛋白與肝細(xì)胞表面的受體蛋白發(fā)生特異性反應(yīng)。CSP和TRAP被認(rèn)為是子孢子入侵肝細(xì)胞的配體蛋白,理由是CSP和TRAP均位于子孢子表面,且它們的抗體能抑制子孢子入侵肝細(xì)胞。CD81分子是表達(dá)于肝細(xì)胞表面的一種膜蛋白,CD81缺陷型小鼠不能感染瘧原蟲。所以CD81可能是肝細(xì)胞表面的受體蛋白。 因此本試驗(yàn)圍繞著惡性瘧原蟲子孢子表面蛋白的免疫原性和子孢子入侵肝細(xì)胞過程中受體和配體的相互作用展開。 根據(jù)惡性瘧原蟲子孢子表面蛋白質(zhì)CSP和TRAP的結(jié)構(gòu)和功能設(shè)計(jì)特異性的引物,將CSP分成3段,分別為C1,C2和C3：將TRAP分成2段,分別為T1和T2。分別構(gòu)建了原核表達(dá)載體pGEX-4T-1-C1, pGEX-4T-1-C2 pGEX-4T-1-C3, pGEX-4T-1-T1和pGEX-4T-1-T2。將上述質(zhì)粒分別轉(zhuǎn)化入BL21-CodonPlus(DE3)-RIPL中。同時根據(jù)CD81大胞外區(qū)(CD81-LEL)的序列設(shè)計(jì)特異性引物,構(gòu)建了原核表達(dá)載體pET22b-CD81-LEL并轉(zhuǎn)化入Rosetta(DE3)中。對鑒定正確的上述重組菌分別應(yīng)用IPTG進(jìn)行誘導(dǎo)表達(dá),經(jīng)SDS-PAGE和Western blot進(jìn)行鑒定,優(yōu)化表達(dá)條件。再分別應(yīng)用Glutathione SepharoseTM 4B和His GraviTrap進(jìn)行純化,經(jīng)過透析除鹽,BCA法進(jìn)行濃度的測定,得到純度和濃度均較高的重組蛋白質(zhì)為研究惡性瘧原蟲子孢子表面蛋白免疫原性和子孢子入侵入肝細(xì)胞的機(jī)制提供物質(zhì)儲備。 在免疫原性分析中采用弗氏佐劑分別與子孢子表面重組蛋白質(zhì)C1, C2 C3, T1和T2混合乳化免疫Wistar大鼠,通過應(yīng)用間接ELISA方法比較不同重組蛋白組中免疫血清的抗體動態(tài)變化、抗體效價(jià)、抗體IgG亞型來確定其免疫原性的強(qiáng)弱。發(fā)現(xiàn)這些子孢子表面蛋白在大鼠體內(nèi)均能產(chǎn)生特異性的抗體,在3免后抗體效價(jià)達(dá)到高點(diǎn),并能持續(xù)一定的時間。所有的子孢子表面蛋白在大鼠內(nèi)產(chǎn)生IgGl/IgG2a的比值均小于1,但接近1。表明以Th1和Th2混合反應(yīng)為主。為惡性瘧原蟲子孢子表面重組蛋白質(zhì)疫苗的研究奠定了基礎(chǔ)。 在入侵機(jī)制研究中將分段表達(dá)純化的惡性瘧原蟲子孢子表面蛋白C1, C2 C3, T1, T2和GST蛋白與人類肝細(xì)胞進(jìn)行黏附試驗(yàn),證實(shí)了CSP和TRAP的各段蛋白具有與HepG2細(xì)胞黏附的特性,其中C3和T2與HepG2細(xì)胞的結(jié)合能力相對較強(qiáng)。將這些蛋白分別與純化的肝細(xì)胞表面蛋白CD81-LEL進(jìn)行結(jié)合試驗(yàn),通過GST-pulldown和His-pulldown證明了重組表達(dá)的子孢子表面蛋白C2與CD81的大胞外區(qū)具有結(jié)合作用。從而在蛋白水平上確定了CD81的大胞外區(qū)與C2蛋白具有結(jié)合能力。該試驗(yàn)為子孢子入侵肝細(xì)胞機(jī)制的研究提供了新的線索。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R382.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 于丹;江鋼鋒;;瘧疾診斷方法研究進(jìn)展[J];華南預(yù)防醫(yī)學(xué);2006年06期

2 李生茂;梁華平;徐祥;劉東擘;;GST Pull-down實(shí)驗(yàn)鑒定NF-κB相互作用多肽[J];免疫學(xué)雜志;2006年01期

3 王英;黃復(fù)生;;CD81分子在瘧原蟲子孢子入侵宿主肝細(xì)胞中的作用[J];熱帶醫(yī)學(xué)雜志;2006年11期

4 許建衛(wèi),鄭汝成,陳愛康,巖松,劉慧,李麗;間接熒光抗體實(shí)驗(yàn)瘧原蟲鏡檢和瘧史訪問結(jié)果間的關(guān)系[J];中國地方病學(xué)雜志;2004年04期

，

本文編號：2045814