约氏疟原虫红外期发育、增殖相关蛋白及YIR蛋白表达与功能研究
发布时间：2018-08-07 08:45
【摘要】： 蚊媒传播的疟疾是一种危害严重的热带传染病,在全世界人群中具有很高的发病率和致死率。近年来疟原虫多药抗性株的出现与迅速扩散,以及蚊媒对杀虫剂耐受性的增加,加之目前又无有效的抗疟疫苗,给疟疾防治工作带来很大的困难。疟疾被认为是复杂寄生虫导致的一种复杂的疾病,由蚊虫叮咬而传播,疟原虫子孢子一旦进入血循环,数分钟后便随血流跨过肝血窦枯否氏细胞(Kupffer cell,KC),主动粘附、迅速侵入肝细胞,进行红外期增殖。然后生成成熟裂殖子侵入红细胞,导致疟疾的发作。红外期指从疟原虫子孢子侵入肝细胞至在肝细胞内发育为成熟的裂殖体然后释放入血液循环前的这段时期,即在肝内的发育时期。早期的研究使人们认识到阻止虫体在肝内发育的疫苗能够有效阻断疟疾的发展。这些研究发现包括:①辐照减毒子孢子免疫动物或人以后,都可以产生长期的消除性免疫,耐受子孢子的攻击。②这些实验模型中的保护性免疫主要由CD4+和CD8+T细胞介导,虽然这并不排除抗体对子孢子所起的作用,却表明了细胞介导的免疫反应对控制肝内原虫的重要性。③流行病学调查也支持由肝细胞内原虫表达的抗原引起的保护性细胞免疫反应所起的作用。 人们一直都努力研究疟原虫各个时期包括红外期的发育、增殖情况。2002年,恶性疟原虫、媒介冈比亚按蚊和约氏疟原虫全基因组测序工作完成,其他种类疟原虫(间日疟原虫、伯氏疟原虫、诺氏疟原虫、鸡疟原虫)也获得了基因组序列的大量数据,为后基因组时代以基因功能分析和蛋白质组学研究为主要任务的研究工作提供了良好的基础。对疟原虫红外期发育相关蛋白与基因的研究,将有助于认识疟原虫生长发育相关的分子机制,从而为疟疾药物和疫苗设计提供新的靶目标,并为采取防治策略战胜疟疾打下坚实的基础。 对人类危害最大的恶性疟原虫,不能进行红外期的体外培养,对其发育情况很难直接进行研究,而宿主为啮齿类的约氏疟原虫是广泛用于子孢子和红外期研究的动物模型。为评价约氏疟原虫与恶性疟原虫间的相互关系,TIGR和海军医学研究中心对约氏疟原虫基因组与恶性疟原虫基因组的数据进行了比较分析,发现约氏疟原虫有一半预测蛋白质与恶性疟原虫直系同源。说明对约氏疟原虫红外期发育、增殖情况的研究,将会在很大程度上帮助我们认识恶性疟原虫这一时期发育的相关情况。 红外期的虫体难以纯化,一方面是数量少,背景高。在各项实验条件理想的情况下,感染情况最好时,也仅有不超过0.3%的肝细胞感染虫体。同时,如何从肝细胞中分离并获取足够数量的虫体是一个棘手的问题,即使应用先进的激光显微捕获切割技术进行分离,其获得的量也相当少,仅适用于进行基因表达分析。这种种困难使得红外期的研究相对于红内期等时期的研究而言进展缓慢,迫切需要有一种较好的方法对红外期的虫体或虫体蛋白进行分离或识别。已知红外期的很多抗原在相近的时期都有表达,例如CSP和TRAP在子孢子时期和红外期都有表达,PyHEP17在红外期及随后的红内期持续表达。因子孢子时期的虫体相对容易获取并进行纯化,这就为我们提供了一个机会:以子孢子的蛋白为抗原免疫动物,获取免疫血清以对红外期的抗原进行识别。本研究即制备了这样的免疫血清,并以此免疫血清识别红外期的虫体蛋白,然后从基因水平分析这些被识别的蛋白,对其中的YIR蛋白进行了原核表达,以期从一个侧面了解疟原虫的发育、增殖过程。实验内容和结果主要包括两个方面: 1.采用免疫印迹与质谱分析相结合的方法,对存在于复杂混合物中的疟原虫红外期相关蛋白进行初步研究。首先分离约氏疟原虫唾液腺子孢子制备成子孢子免疫抗原,程序免疫新西兰大白兔,获取免疫血清。同时,以成熟的唾液腺子孢子经尾静脉注射感染Sprague Dawley (SD)大鼠,感染后43h取大鼠肝脏,供提取感染大鼠肝脏总蛋白,同时HE染色观察感染情况。以此提取的蛋白进行SDS-PAGE电泳,以获取的免疫血清为一抗进行Western Blotting分析,结果成功地识别出疟原虫的相关蛋白条带。切取与NC膜上识别出的条带对应的凝胶条带上的蛋白进行飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time Of Flying Mass Spectrometry,MALDI-TOF-MS)和二级质谱(Liquid Chromatography/Mass Spectrometry,LC/MS)分析,获得疟原虫相关的蛋白的信息。获得初步结果后,为进一步确定其中的蛋白特性,将提取的感染肝脏蛋白进行了双向电泳及Western Blotting分析,确定目标蛋白点,进行飞行时间质谱分析。对质谱结果的生物信息学分析,获得了有意义的结果,即发现YIR蛋白在红外期有表达。而以前对YIR蛋白的研究都集中在红内期,认为其表达的高度变异与疟原虫逃避宿主的免疫相关。在红外期多核裂殖体中发现此蛋白表达,一方面说明此蛋白在疟原虫中的出现是一个连续的过程,并不与虫体的分期完全平行;另一方面也提示,对研究难度大的红外期,可以继续采用此方法,以其他多个时期,如红内期,卵囊子孢子时期等的免疫血清进行识别,并进行进一步的研究,寻找相关的蛋白。 2.RT-PCR扩增yir基因,以pQE80L表达质粒为基础构建原核表达质粒pQE80L/YIR,并在DH5α宿主菌中表达YIR蛋白。质谱的结果使本实验研究的对象从几千上万种蛋白聚焦到其中的单个蛋白,对这单个分子的研究主要从基因和蛋白两个水平进行。根据质谱分析得到的结果,参考文献,设计特异引物,以RT-PCR从约氏疟原虫感染后43h的大鼠肝脏中成功地扩增出yir基因片段,将PCR产物TA克隆到pMD-18 Simple载体中,进行测序。测序结果在NCBI,TIGR上进行BLAST检索,确定得到的基因属于yir基因家族,证实了第一阶段蛋白研究的结果。为了进一步研究yir,利用原核表达系统对扩增出的yir基因进行了表达。选择pQE80L为表达载体,DH5α为宿主菌,在yir基因片段的两端添加酶切位点后将此基因片段定向克隆到表达载体中,IPTG诱导表达YIR蛋白,并以免疫印迹成功地检测到了此蛋白的表达。一方面验证了质谱分析的结果,另一方面为下一步深入研究yir/YIR在红外期的增殖发育过程中的作用研究奠定了基础。 综上所述,本实验通过免疫印迹的方法结合2-DE与生物质谱技术,克服了红外期的低感染率与高背景,直接对研究难度大的红外期蛋白进行研究,首次发现YIR蛋白在红外期的表达。根据蛋白研究的结果,以RT-PCR对相关的分子进行了扩增,发现了yir基因在红外期的表达,从基因水平证实了YIR蛋白的表达,成功构建了YIR蛋白的表达载体,表达了YIR蛋白。这些基础性的工作为为下一步阐明红外期疟原虫的增殖、发育相关的分子变化提供基础。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383
本文编号：2169505
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383
【参考文献】
相关期刊论文 前2条
1 高会广,何凤田,李蓉芬,陈珊,唐蓓,彭家和,陈敏,周度金;人BAFF cDNA的克隆及原核表达载体的构建[J];第三军医大学学报;2003年06期
2 杨清浩,王祥卫,金燕,张立新;MUC1抗原的B细胞表位预测[J];第三军医大学学报;2005年05期
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