新型转基因减毒疟疾疫苗的构建与抗疟机理研究

发布时间：2018-07-05 00:54

本文选题：B淋巴细胞趋化因子 + 疟疾　；参考：《海南大学》2011年博士论文

【摘要】：疟疾是一种古老的寄生虫传染病,严重威胁着人类健康,阻碍着社会发展。据WHO最新公布数字,全球有一半人口受到疟疾的威胁,2008年有2.43亿人感染疟原虫,约86.3万人因患疟疾死亡,大多是5岁以下的儿童。近年来,虽然在疟疾控制方面取得了显著的成绩,但由于疟原虫抗药性以及蚊媒对杀虫剂抗性的产生和扩散,使疟疾防治面临严重困难。当前普遍认为,研制安全、价廉和有效的疫苗是人类控制乃至根除疟疾的重要途径。世界卫生组织、联合国计划开发署、世界银行等已将疟疾疫苗研究项目作为全球优先发展的三大疫苗项目之一。疟疾感染从雌性按蚊叮咬人体,其唾液腺中的成熟疟原虫子孢子随蚊虫的唾液注入宿主血液循环开始,随后子孢子进入肝细胞,在其中行裂体增殖而形成裂殖体。成熟后肝细胞破裂,裂殖子被释放入血液,进入红细胞并在其中行裂体增殖。经过若干增殖周期后致使红细胞破裂,出现临床症状。由此可见,子孢子是疟疾感染人类宿主的上游环节,以子孢子作为疟疾疫苗引起有效的免疫应答可阻断子孢子进入肝细胞从而终止疟疾生活史,达到有效防治疟疾的目的。疟疾疫苗的开发研究已有40多年的历史,经历了全虫疫苗(包括死疫苗和放射减毒活疫苗)、基因工程亚单位疫苗和化学合成的多肽疫苗、核酸及病原载体疫苗等几个时期。但是,所有这些疫苗在人体试验中都没有达到理想的效果,其中疫苗的安全性、诱导的免疫应答特异性和效价不高、免疫力持续时间短等都是其中原因之一。如何兼顾疫苗的安全性及增高疟疾疫苗的免疫反应能力,延长疫苗免疫反应的时间是疟疾研究疫苗研究的重要内容。 Mueller等人用基因敲除的方法将疟原虫UIS3基因敲除后,发现缺失了UIS3基因的子孢子具有减毒功能,感染动物模型后疟原虫子孢子停留于肝细胞期而不发展为红细胞期疟疾而终止疟疾生活史,将其作为减毒全虫疫苗,在小鼠疟疾模型中可以诱导产生十分理想的免疫保护作用。本研究利用基因打靶技术敲除伯氏疟原虫的UIS3基因,同时为了获得良好的免疫效果,利用转基因技术将具有免疫增强作用的B淋巴细胞趋化因子(B-lymphocytechemoattractant,BLC)转进基因工程减毒的伯氏疟原虫UIS3基因敲除质粒中再转染疟原虫子孢子,以期获得一种既有减毒,又能够增加免疫功能的新型疟原虫株(UIS3-/BLC+)。 BLC是近年新发现的一种特异性趋化B细胞的细胞因子,并能刺激细胞表达趋化因子受体BLR1,参与引导B细胞的游走和归巢,能吸引B细胞和CD4+T细胞趋化到免疫应答部位从而增强特异免疫应答。将BLC基因修饰疟疾疫苗,则可增强针对疟原虫的特异性体液免疫应答及细胞免疫应答。本研究根据基因库提供的小鼠BLC基因序列设计克隆扩增BLC的cDNA基因序列的引物,利用RT-PCR技术获得小鼠BLC cDNA基因,通过限制性内切酶酶切和T4酶连接,将BLC基因cDNA序列插入伯氏疟原虫UIS3基因敲除质粒中,获得重组伯氏疟原虫BLC转基因重组质粒。重组质粒含有息疟定抗性基因和绿色荧光蛋白的报告基因,可在转化子中表达。筛选重组质粒并经酶切和DNA序列测定证实正确性后,体外转染真核细胞。经琼脂糖凝胶电泳发现扩增的小鼠BLC基因cDNA序列分子量大小和预期值相一致,重组质粒酶切结果和预期值相符,DNA序列测定显示重组质粒的目的基因阅读框架准确无误。从mRNA蛋白质表达二个层面证明重组质粒可以在真核细胞中有效表达BLC基因。证明已成功构建了伯氏疟原虫BLC转基因重组质粒(UIS3-/BLC+)。伯氏疟原虫在转化前经体外短期培养,利用密度梯度介质Renografin分离子孢子用于电穿孔转化。将构建的伯氏疟原虫BLC转基因重组质粒经酶切线性化后转染伯氏疟原虫,获得UIS3-/BLC+疟原虫株。将此疟原虫株经尾静脉注入小鼠体内,用息疟定腹腔内注射进行药物筛选,经2轮筛选后,得到了阳性的伯氏疟原虫转化子。在荧光显微镜下观察到经息疟定筛选的原虫呈现绿色荧光；经PCR检测到了重组质粒的存在,说明重组质粒已正确的整合在伯氏疟原虫基因之中。本研究进一步在小鼠伯氏疟疾模型中,采用分子生物学、免疫学和组织学等技术对获得的UIS3-/BLC+疟原虫株进行抗疟机理研究。结果发现UIS3-/BLC+疟原虫株可以在肝细胞期有效生长,但未能有效发育转化为红内期,具备了基因敲除减毒的特性。与UIS3子孢子免疫相比,我们发现,UIS3-/BLC+子孢子免疫的小鼠可以有效产生体液和细胞免疫反应,抗体的水平和持续时间均明显强于UIS3-子孢子,T淋巴细胞特异杀伤疟原虫的能力也明显增强。体内外抗疟疾感染试验结果也表明,UIS3-/BLC+子孢子作为疫苗较UIS3-具有更好的抑制疟原虫生长和转化的作用。本研究结果表明,基因工程敲除并转入免疫增强基因的UIS3-/BLC+疟原虫子孢子是一种很好的疟疾疫苗模式,具有较好的临床应用前景,值得深入研究。
[Abstract]:Malaria is an ancient parasitic infectious disease, which is a serious threat to human health and hinders social development. According to the latest WHO figures, half of the world's population is threatened by malaria. In 2008, 243 million people were infected with malaria parasites, about 863 thousand people died of malaria, mostly children under 5 years of age. In recent years, in spite of malaria control, Significant achievements have been achieved, but the prevention and control of malaria is seriously difficult due to the resistance of malaria parasites and the generation and diffusion of mosquito vector resistance to insecticides. It is widely believed that the development of safe, inexpensive and effective vaccines is an important way for human control and eradication of malaria. The WHO, the United Nations Programme Development Agency, the world bank, etc. Malaria vaccine research project is one of the three priority vaccine projects in the world.
The malaria infection from the female Anopheles mosquito bites human body, the mature Plasmodium sporozoite in its salivary gland begins with the injection of the mosquito's saliva into the host blood circulation, then the sporozoite enters the liver cell and forms a fissure body in which the fissure body is proliferated, and the liver cells break up, the merozoites are released into the blood, into the red cells and increase in the fissure body. It can be seen that subspore is the upper link of malaria infection in the human host, and the effective immune response of the subspore as a malaria vaccine can block the subspore into the liver cells and terminate the history of malaria, and achieve the purpose of effective prevention and control of malaria.
The development and research of malaria vaccine has been developed for more than 40 years. It has experienced the whole insect vaccine (including dead vaccine and live attenuated vaccine), genetic engineering subunit vaccine and chemical synthetic peptide vaccine, nucleic acid and pathogen carrier vaccine. However, all these vaccines have not achieved the ideal effect in human trials, among them, the epidemic vaccine is not satisfactory. The safety of the vaccine, the induced immune response specificity and the low titer and the short duration of the immunity are one of the reasons. How to take into account the safety of the vaccine and increase the immune response ability of the vaccine and prolong the time of the immune response of the vaccine are the important contents of the Research on the vaccine of malaria.
Mueller and others knocked out the Plasmodium UIS3 gene with gene knockout, and found that the sporozoite missing the UIS3 gene had the detoxification function. After infection of the animal model, the Plasmodium sporozoite stayed at the liver cell stage and did not develop to the red cell stage malaria and terminated the malaria life history. It was used as a attenuated total worm vaccine and in the model of the mice malaria. In this study, the gene targeting technique was used to knock out the UIS3 gene of Plasmodium Bergh, and in order to obtain good immune effect, the B lymphocyte chemoattractant factor (B-lymphocytechemoattractant, BLC) with immune enhancement was transferred into the gene engineering attenuated herb. Plasmodium UIS3 gene knockout plasmids transfected into the Plasmodium sporozoite to obtain a new type of Plasmodium (UIS3-/BLC+), which can reduce the toxicity and increase the immune function.
BLC is a newly discovered cytokine that specifically chemotaxis B cells in recent years, and stimulates cell expression of chemokine receptor BLR1, and participates in guiding the walking and homing of B cells. It can attract B cells and CD4+T cells to chemotaxis to the immune response site and thus enhance the specific immune response. The modification of the BLC gene to malaria vaccine can enhance the malaria parasite. Specific humoral immune response and cellular immune response.
In this study, the primers of the cDNA gene sequence of BLC were cloned according to the gene sequence of the mouse BLC gene provided by the gene bank. The BLC cDNA gene of mice was obtained by RT-PCR technique. The cDNA sequence of BLC gene was inserted into the UIS3 gene knockout plasmid of Plasmodium bergpari by restriction endonuclease digestion and T4 enzyme connection. The recombinant plasmid of recombinant Plasmodium bergpari was obtained. Recombinant plasmid containing the reporter gene of the Plasmodium resistance gene and green fluorescent protein can be expressed in the transformant. After screening the recombinant plasmid and confirmed by enzyme digestion and DNA sequence, eukaryotic cells were transfected in vitro. The molecular weight and expectation of the cDNA sequence of the amplified BLC gene were detected by agarose gel electrophoresis. The results of the recombinant plasmids were consistent with the expected values. DNA sequencing showed that the target gene reading frame of the recombinant plasmid was accurate. The recombinant plasmid could express the BLC gene effectively in eukaryotic cells from the two levels of mRNA protein expression. It proved that the recombinant plasmid of Plasmodium bergi BLC transgenic plasmid has been successfully constructed (UIS3-/BL C+).
The Plasmodium Bergh was cultured in vitro and used in short term culture in vitro to use the density gradient medium Renografin ion spore for electroporation. The Plasmodium berberi BLC transgenic plasmid was transfected into Plasmodium Bergh and the Plasmodium UIS3-/BLC+ was transfected to the Plasmodium bergpari. After 2 rounds of screening, the positive Plasmodium berberi transformant was obtained after 2 rounds of screening. The parasites screened by the fluorescence microscope showed green fluorescence under the fluorescence microscope, and the recombinant plasmids were detected by the recombinant plasmids, which showed that the recombinant plasmid was correctly integrated in the Plasmodium bergpari gene.
In this study, we further studied the antimalarial mechanism of the UIS3-/BLC+ Plasmodium Plasmodium strain obtained by molecular biology, immunology and histology in the BBER's malaria model in mice. The results showed that the Plasmodium UIS3-/BLC+ could grow effectively in the hepatocyte stage, but failed to effectively develop into the red period, and had a gene knockout attenuated strain. Compared with the UIS3 subspore immunization, we found that the mice immunized with UIS3-/BLC+ sporozoites could effectively produce humoral and cellular immune responses. The level and duration of the antibody were significantly stronger than the UIS3- sporozoites, and the ability of T lymphocytes to kill the Plasmodium specifically was also enhanced. The results of anti malaria infection test in the body and outside of the body also showed that UIS3- /BLC+ sporozoite as a vaccine has better inhibitory effect on growth and transformation of malaria than UIS3-.
The results of this study show that the UIS3-/BLC+ Plasmodium sporozoites, which have been knocked out by gene engineering and transferred into the immune enhancement gene, are a good model of malaria vaccine. It has a good prospect for clinical application and is worthy of further study.
【学位授予单位】：海南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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