【摘要】： 蚊媒傳播的瘧疾是一種危害嚴(yán)重的熱帶傳染病,在全世界人群中具有很高的發(fā)病率和致死率。近年來瘧原蟲多藥抗性株的出現(xiàn)與迅速擴(kuò)散,以及蚊媒對(duì)殺蟲劑耐受性的增加,加之目前又無有效的抗瘧疫苗,給瘧疾防治工作帶來很大的困難。瘧疾被認(rèn)為是復(fù)雜寄生蟲導(dǎo)致的一種復(fù)雜的疾病,由蚊蟲叮咬而傳播,瘧原蟲子孢子一旦進(jìn)入血循環(huán),數(shù)分鐘后便隨血流跨過肝血竇枯否氏細(xì)胞(Kupffer cell,KC),主動(dòng)粘附、迅速侵入肝細(xì)胞,進(jìn)行紅外期增殖。然后生成成熟裂殖子侵入紅細(xì)胞,導(dǎo)致瘧疾的發(fā)作。紅外期指從瘧原蟲子孢子侵入肝細(xì)胞至在肝細(xì)胞內(nèi)發(fā)育為成熟的裂殖體然后釋放入血液循環(huán)前的這段時(shí)期,即在肝內(nèi)的發(fā)育時(shí)期。早期的研究使人們認(rèn)識(shí)到阻止蟲體在肝內(nèi)發(fā)育的疫苗能夠有效阻斷瘧疾的發(fā)展。這些研究發(fā)現(xiàn)包括:①輻照減毒子孢子免疫動(dòng)物或人以后,都可以產(chǎn)生長期的消除性免疫,耐受子孢子的攻擊。②這些實(shí)驗(yàn)?zāi)Ｐ椭械谋Ｗo(hù)性免疫主要由CD4+和CD8+T細(xì)胞介導(dǎo),雖然這并不排除抗體對(duì)子孢子所起的作用,卻表明了細(xì)胞介導(dǎo)的免疫反應(yīng)對(duì)控制肝內(nèi)原蟲的重要性。③流行病學(xué)調(diào)查也支持由肝細(xì)胞內(nèi)原蟲表達(dá)的抗原引起的保護(hù)性細(xì)胞免疫反應(yīng)所起的作用。 人們一直都努力研究瘧原蟲各個(gè)時(shí)期包括紅外期的發(fā)育、增殖情況。2002年,惡性瘧原蟲、媒介岡比亞按蚊和約氏瘧原蟲全基因組測(cè)序工作完成,其他種類瘧原蟲(間日瘧原蟲、伯氏瘧原蟲、諾氏瘧原蟲、雞瘧原蟲)也獲得了基因組序列的大量數(shù)據(jù),為后基因組時(shí)代以基因功能分析和蛋白質(zhì)組學(xué)研究為主要任務(wù)的研究工作提供了良好的基礎(chǔ)。對(duì)瘧原蟲紅外期發(fā)育相關(guān)蛋白與基因的研究,將有助于認(rèn)識(shí)瘧原蟲生長發(fā)育相關(guān)的分子機(jī)制,從而為瘧疾藥物和疫苗設(shè)計(jì)提供新的靶目標(biāo),并為采取防治策略戰(zhàn)勝瘧疾打下堅(jiān)實(shí)的基礎(chǔ)。 對(duì)人類危害最大的惡性瘧原蟲,不能進(jìn)行紅外期的體外培養(yǎng),對(duì)其發(fā)育情況很難直接進(jìn)行研究,而宿主為嚙齒類的約氏瘧原蟲是廣泛用于子孢子和紅外期研究的動(dòng)物模型。為評(píng)價(jià)約氏瘧原蟲與惡性瘧原蟲間的相互關(guān)系,TIGR和海軍醫(yī)學(xué)研究中心對(duì)約氏瘧原蟲基因組與惡性瘧原蟲基因組的數(shù)據(jù)進(jìn)行了比較分析,發(fā)現(xiàn)約氏瘧原蟲有一半預(yù)測(cè)蛋白質(zhì)與惡性瘧原蟲直系同源。說明對(duì)約氏瘧原蟲紅外期發(fā)育、增殖情況的研究,將會(huì)在很大程度上幫助我們認(rèn)識(shí)惡性瘧原蟲這一時(shí)期發(fā)育的相關(guān)情況。 紅外期的蟲體難以純化,一方面是數(shù)量少,背景高。在各項(xiàng)實(shí)驗(yàn)條件理想的情況下,感染情況最好時(shí),也僅有不超過0.3%的肝細(xì)胞感染蟲體。同時(shí),如何從肝細(xì)胞中分離并獲取足夠數(shù)量的蟲體是一個(gè)棘手的問題,即使應(yīng)用先進(jìn)的激光顯微捕獲切割技術(shù)進(jìn)行分離,其獲得的量也相當(dāng)少,僅適用于進(jìn)行基因表達(dá)分析。這種種困難使得紅外期的研究相對(duì)于紅內(nèi)期等時(shí)期的研究而言進(jìn)展緩慢,迫切需要有一種較好的方法對(duì)紅外期的蟲體或蟲體蛋白進(jìn)行分離或識(shí)別。已知紅外期的很多抗原在相近的時(shí)期都有表達(dá),例如CSP和TRAP在子孢子時(shí)期和紅外期都有表達(dá),PyHEP17在紅外期及隨后的紅內(nèi)期持續(xù)表達(dá)。因子孢子時(shí)期的蟲體相對(duì)容易獲取并進(jìn)行純化,這就為我們提供了一個(gè)機(jī)會(huì):以子孢子的蛋白為抗原免疫動(dòng)物,獲取免疫血清以對(duì)紅外期的抗原進(jìn)行識(shí)別。本研究即制備了這樣的免疫血清,并以此免疫血清識(shí)別紅外期的蟲體蛋白,然后從基因水平分析這些被識(shí)別的蛋白,對(duì)其中的YIR蛋白進(jìn)行了原核表達(dá),以期從一個(gè)側(cè)面了解瘧原蟲的發(fā)育、增殖過程。實(shí)驗(yàn)內(nèi)容和結(jié)果主要包括兩個(gè)方面: 1.采用免疫印跡與質(zhì)譜分析相結(jié)合的方法,對(duì)存在于復(fù)雜混合物中的瘧原蟲紅外期相關(guān)蛋白進(jìn)行初步研究。首先分離約氏瘧原蟲唾液腺子孢子制備成子孢子免疫抗原,程序免疫新西蘭大白兔,獲取免疫血清。同時(shí),以成熟的唾液腺子孢子經(jīng)尾靜脈注射感染Sprague Dawley (SD)大鼠,感染后43h取大鼠肝臟,供提取感染大鼠肝臟總蛋白,同時(shí)HE染色觀察感染情況。以此提取的蛋白進(jìn)行SDS-PAGE電泳,以獲取的免疫血清為一抗進(jìn)行Western Blotting分析,結(jié)果成功地識(shí)別出瘧原蟲的相關(guān)蛋白條帶。切取與NC膜上識(shí)別出的條帶對(duì)應(yīng)的凝膠條帶上的蛋白進(jìn)行飛行時(shí)間質(zhì)譜(Matrix-Assisted Laser Desorption/Ionization Time Of Flying Mass Spectrometry,MALDI-TOF-MS)和二級(jí)質(zhì)譜(Liquid Chromatography/Mass Spectrometry,LC/MS)分析,獲得瘧原蟲相關(guān)的蛋白的信息。獲得初步結(jié)果后,為進(jìn)一步確定其中的蛋白特性,將提取的感染肝臟蛋白進(jìn)行了雙向電泳及Western Blotting分析,確定目標(biāo)蛋白點(diǎn),進(jìn)行飛行時(shí)間質(zhì)譜分析。對(duì)質(zhì)譜結(jié)果的生物信息學(xué)分析,獲得了有意義的結(jié)果,即發(fā)現(xiàn)YIR蛋白在紅外期有表達(dá)。而以前對(duì)YIR蛋白的研究都集中在紅內(nèi)期,認(rèn)為其表達(dá)的高度變異與瘧原蟲逃避宿主的免疫相關(guān)。在紅外期多核裂殖體中發(fā)現(xiàn)此蛋白表達(dá),一方面說明此蛋白在瘧原蟲中的出現(xiàn)是一個(gè)連續(xù)的過程,并不與蟲體的分期完全平行;另一方面也提示,對(duì)研究難度大的紅外期,可以繼續(xù)采用此方法,以其他多個(gè)時(shí)期,如紅內(nèi)期,卵囊子孢子時(shí)期等的免疫血清進(jìn)行識(shí)別,并進(jìn)行進(jìn)一步的研究,尋找相關(guān)的蛋白。 2.RT-PCR擴(kuò)增yir基因,以pQE80L表達(dá)質(zhì)粒為基礎(chǔ)構(gòu)建原核表達(dá)質(zhì)粒pQE80L/YIR,并在DH5α宿主菌中表達(dá)YIR蛋白。質(zhì)譜的結(jié)果使本實(shí)驗(yàn)研究的對(duì)象從幾千上萬種蛋白聚焦到其中的單個(gè)蛋白,對(duì)這單個(gè)分子的研究主要從基因和蛋白兩個(gè)水平進(jìn)行。根據(jù)質(zhì)譜分析得到的結(jié)果,參考文獻(xiàn),設(shè)計(jì)特異引物,以RT-PCR從約氏瘧原蟲感染后43h的大鼠肝臟中成功地?cái)U(kuò)增出yir基因片段,將PCR產(chǎn)物TA克隆到pMD-18 Simple載體中,進(jìn)行測(cè)序。測(cè)序結(jié)果在NCBI,TIGR上進(jìn)行BLAST檢索,確定得到的基因?qū)儆趛ir基因家族,證實(shí)了第一階段蛋白研究的結(jié)果。為了進(jìn)一步研究yir,利用原核表達(dá)系統(tǒng)對(duì)擴(kuò)增出的yir基因進(jìn)行了表達(dá)。選擇pQE80L為表達(dá)載體,DH5α為宿主菌,在yir基因片段的兩端添加酶切位點(diǎn)后將此基因片段定向克隆到表達(dá)載體中,IPTG誘導(dǎo)表達(dá)YIR蛋白,并以免疫印跡成功地檢測(cè)到了此蛋白的表達(dá)。一方面驗(yàn)證了質(zhì)譜分析的結(jié)果,另一方面為下一步深入研究yir/YIR在紅外期的增殖發(fā)育過程中的作用研究奠定了基礎(chǔ)。 綜上所述,本實(shí)驗(yàn)通過免疫印跡的方法結(jié)合2-DE與生物質(zhì)譜技術(shù),克服了紅外期的低感染率與高背景,直接對(duì)研究難度大的紅外期蛋白進(jìn)行研究,首次發(fā)現(xiàn)YIR蛋白在紅外期的表達(dá)。根據(jù)蛋白研究的結(jié)果,以RT-PCR對(duì)相關(guān)的分子進(jìn)行了擴(kuò)增,發(fā)現(xiàn)了yir基因在紅外期的表達(dá),從基因水平證實(shí)了YIR蛋白的表達(dá),成功構(gòu)建了YIR蛋白的表達(dá)載體,表達(dá)了YIR蛋白。這些基礎(chǔ)性的工作為為下一步闡明紅外期瘧原蟲的增殖、發(fā)育相關(guān)的分子變化提供基礎(chǔ)。
[Abstract]:Mosquito borne malaria is a serious tropical infectious disease, which has high morbidity and mortality worldwide. In recent years, the emergence and rapid spread of multidrug resistant strains of Plasmodium, as well as the increase of mosquito resistance to insecticides, and the lack of effective antimalarial vaccines at present, have brought great difficulties to the prevention and control of malaria. Malaria is considered to be a complex disease caused by the complex parasite, which is transmitted by mosquito bites. Once the Plasmodium sporozoite enters the blood circulation, after a few minutes, it crosses the liver blood sinus Kupffer cells (Kupffer cell, KC), invades the liver cells rapidly and proliferates in the infrared period, and then produces mature merozoites to invade red. Cells, causing the attack of malaria. The infrared period refers to the period from the Plasmodium sporozoite invasion of the liver cells to the mature fissure in the liver cells and then released into the blood circulation, that is, in the period of development in the liver. Early studies have made people realize that the vaccine that prevents the growth of the insect body in the liver can effectively block the development of malaria. These findings include: (1) irradiated detoxification subspore immune animals or humans can produce long-term elimination of immunity and tolerate subspore attacks. 2. Protective immunity in these experimental models is mediated mainly by CD4+ and CD8+T cells, although this does not exclude the effect of antibodies against sporozoites, but it indicates cell mediated The importance of immune response to the control of intrahepatic protozoa. (3) epidemiological surveys also support the role of protective cellular immune responses caused by antigens expressed by protozoa in hepatocytes.
People have been working hard to study the development of the Plasmodium, including the infrared period, the proliferation of.2002 years, Plasmodium falciparum, the vector of Anopheles Gambia and Plasmodium. The other species of Plasmodium vivax (Plasmodium vivax, Plasmodium bergpari, Plasmodium Nobel, and Plasmodium) also obtained a large number of genome sequences. The data provide a good basis for the research work of the post genome era with genetic functional analysis and proteomics research. The study of Plasmodium IR developmental related proteins and genes will help to understand the molecular mechanisms related to the growth and development of Plasmodium, thus providing new targets for the design of malaria drugs and vaccines. Objective and lay a solid foundation for the prevention and cure of malaria.
The most endangered species of Plasmodium falciparum can not be cultured in vitro, and it is difficult to study its development. The host of rodent Plasmodium falciparum is an animal model widely used in the study of sporozoite and infrared phase. To evaluate the relationship between Plasmodium and malarial parasites, TIGR and Naval Medicine A comparative analysis of the genome of Plasmodium John's Plasmodium falciparum and the genome of Plasmodium falciparum showed that half of the Plasmodium falciparum predicted the direct homology of Plasmodium falciparum to the Plasmodium falciparum. The development of the period.
In the infrared phase, the insect body is difficult to purify. On the one hand, the number is small and the background is high. When the conditions are ideal, the infection is best and only 0.3% of the liver cells are infected. At the same time, it is a difficult problem to separate and obtain sufficient number of worms from the liver cells, even if the advanced laser microcapture is used. This kind of difficulty makes the research of the infrared period relatively slow in the study of the period of the red period and so on. There is an urgent need for a better method to separate or identify the insect body or the insect body protein in the infrared period. Many antigens are expressed at the same time, such as the expression of CSP and TRAP in the sporozoite and infrared periods. PyHEP17 is expressed continuously in the infrared period and in the subsequent red period. The body of the factor spore period is relatively easy to obtain and purify. This provides us with an opportunity to immunize the animals with the subspore protein as an antigen. The immune sera was taken to identify the antigen of the infrared phase. The immune sera was prepared and the immune sera was used to identify the insect body protein of the infrared period. Then the identified proteins were analyzed from the gene level and the YIR protein was expressed in the prokaryotic expression to understand the development of the Plasmodium from one side. The contents and results of the experiment mainly include two aspects:
1. the method of immunoblotting and mass spectrometry was used to study the infrared phase proteins of Plasmodium Plasmodium in complex mixtures. First, the immuno antigens were isolated from the spore of the salivary glands of Plasmodium falciparum, and the New Zealand white rabbits were immunized to obtain the immune sera. The rats infected with Sprague Dawley (SD) were injected into the tail vein, and the liver was extracted from the rat 43h after infection. The infected rat liver protein was extracted and the infection was observed by HE staining. The extracted protein was obtained by SDS-PAGE electrophoresis, and the immune sera was obtained by Western Blotting analysis. The results were successfully identified as the related eggs of the Plasmodium. The white strip. Cut the protein on the gel strip corresponding to the strip identified by the NC membrane for the time of flight mass spectrometry (Matrix-Assisted Laser Desorption/Ionization Time Of Flying Mass Spectrometry, MALDI-TOF-MS) and the two level mass spectrometry (Liquid Chromatography/Mass) analysis to obtain the proteins related to the Plasmodium After obtaining the preliminary results, in order to further determine the protein characteristics of the protein, the extracted infected liver proteins were analyzed by two-dimensional electrophoresis and Western Blotting, the target protein points were determined and the time of flight mass spectrometry was analyzed. The bioinformatics analysis of the results of the mass spectrometry obtained a meaningful result, that is, the YIR protein was found in the infrared period. The previous study of YIR protein was concentrated in the red period, and it was considered that the high variation of the expression was related to the immunity of the parasite to escape the host immunity. The protein expression was found in the infrared multicore fission body. On the one hand, the appearance of the protein in the Plasmodium was a continuous process, and the other was not completely parallel to the staging of the insect body; It is also suggested that this method can be used to identify immune sera from other periods, such as the red period and the oocyst spores, and to further study the related proteins.
The YiR gene was amplified by 2.RT-PCR, and the prokaryotic expression plasmid pQE80L/YIR was constructed on the basis of pQE80L expression plasmid, and YIR protein was expressed in DH5 alpha host bacteria. The results of mass spectrometry made the object of this study focus from thousands of proteins to one of the single proteins. The research on this single molecule was mainly carried out from two levels of gene and protein. According to the results obtained by the mass spectrometry analysis, specific primers were designed and the YiR gene fragment was successfully amplified from the liver of 43h rats infected with Plasmodium Joshua. The PCR product TA was cloned into the pMD-18 Simple vector and sequenced. The sequence was sequenced in NCBI and TIGR, and the gene was determined to be a YiR geneticist. In order to further study the results of the first stage protein study, in order to further study YiR, the amplified YiR gene was expressed by the prokaryotic expression system. PQE80L was selected as the expression vector, DH5 alpha was the host bacteria, and the fragment was cloned into the expression vector by adding the enzyme cut site at the two ends of the YiR gene fragment, and IPTG induced the expression of YIR. The protein was successfully detected by immunoblotting. On the one hand, the results of mass spectrometry analysis were verified. On the other hand, the basis for further research on the role of yir/YIR during the proliferation and development of the infrared phase was laid.
To sum up, this experiment overcame the low infection rate and high background of the infrared phase by using the method of immunoblotting combined with 2-DE and biological mass spectrometry. It was the first time to study the difficult infrared phase protein and the first discovery of the expression of YIR protein in the infrared period. According to the results of the protein study, the related molecules were amplified by RT-PCR. The expression of the YiR gene in the infrared period was presented. The expression of YIR protein was confirmed at the gene level. The expression vector of YIR protein was successfully constructed and the YIR protein was expressed. These basic work provided the basis for clarifying the proliferation of the malaria parasite in the infrared phase and the molecular changes related to development next.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R383

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本文編號(hào)：2169505