發(fā)布時間：2018-07-05 00:54

  本文選題：B淋巴細胞趨化因子 + 瘧疾��； 參考：《海南大學(xué)》2011年博士論文

【摘要】：瘧疾是一種古老的寄生蟲傳染病,嚴重威脅著人類健康,阻礙著社會發(fā)展。據(jù)WHO最新公布數(shù)字,全球有一半人口受到瘧疾的威脅,2008年有2.43億人感染瘧原蟲,約86.3萬人因患瘧疾死亡,大多是5歲以下的兒童。近年來,雖然在瘧疾控制方面取得了顯著的成績,但由于瘧原蟲抗藥性以及蚊媒對殺蟲劑抗性的產(chǎn)生和擴散,使瘧疾防治面臨嚴重困難。當(dāng)前普遍認為,研制安全、價廉和有效的疫苗是人類控制乃至根除瘧疾的重要途徑。世界衛(wèi)生組織、聯(lián)合國計劃開發(fā)署、世界銀行等已將瘧疾疫苗研究項目作為全球優(yōu)先發(fā)展的三大疫苗項目之一。 瘧疾感染從雌性按蚊叮咬人體,其唾液腺中的成熟瘧原蟲子孢子隨蚊蟲的唾液注入宿主血液循環(huán)開始,隨后子孢子進入肝細胞,在其中行裂體增殖而形成裂殖體。成熟后肝細胞破裂,裂殖子被釋放入血液,進入紅細胞并在其中行裂體增殖。經(jīng)過若干增殖周期后致使紅細胞破裂,出現(xiàn)臨床癥狀。由此可見,子孢子是瘧疾感染人類宿主的上游環(huán)節(jié),以子孢子作為瘧疾疫苗引起有效的免疫應(yīng)答可阻斷子孢子進入肝細胞從而終止瘧疾生活史,達到有效防治瘧疾的目的。 瘧疾疫苗的開發(fā)研究已有40多年的歷史,經(jīng)歷了全蟲疫苗(包括死疫苗和放射減毒活疫苗)、基因工程亞單位疫苗和化學(xué)合成的多肽疫苗、核酸及病原載體疫苗等幾個時期。但是,所有這些疫苗在人體試驗中都沒有達到理想的效果,其中疫苗的安全性、誘導(dǎo)的免疫應(yīng)答特異性和效價不高、免疫力持續(xù)時間短等都是其中原因之一。如何兼顧疫苗的安全性及增高瘧疾疫苗的免疫反應(yīng)能力,延長疫苗免疫反應(yīng)的時間是瘧疾研究疫苗研究的重要內(nèi)容。 Mueller等人用基因敲除的方法將瘧原蟲UIS3基因敲除后,發(fā)現(xiàn)缺失了UIS3基因的子孢子具有減毒功能,感染動物模型后瘧原蟲子孢子停留于肝細胞期而不發(fā)展為紅細胞期瘧疾而終止瘧疾生活史,將其作為減毒全蟲疫苗,在小鼠瘧疾模型中可以誘導(dǎo)產(chǎn)生十分理想的免疫保護作用。本研究利用基因打靶技術(shù)敲除伯氏瘧原蟲的UIS3基因,同時為了獲得良好的免疫效果,利用轉(zhuǎn)基因技術(shù)將具有免疫增強作用的B淋巴細胞趨化因子(B-lymphocytechemoattractant,BLC)轉(zhuǎn)進基因工程減毒的伯氏瘧原蟲UIS3基因敲除質(zhì)粒中再轉(zhuǎn)染瘧原蟲子孢子,以期獲得一種既有減毒,又能夠增加免疫功能的新型瘧原蟲株(UIS3-/BLC+)。 BLC是近年新發(fā)現(xiàn)的一種特異性趨化B細胞的細胞因子,并能刺激細胞表達趨化因子受體BLR1,參與引導(dǎo)B細胞的游走和歸巢,能吸引B細胞和CD4+T細胞趨化到免疫應(yīng)答部位從而增強特異免疫應(yīng)答。將BLC基因修飾瘧疾疫苗,則可增強針對瘧原蟲的特異性體液免疫應(yīng)答及細胞免疫應(yīng)答。 本研究根據(jù)基因庫提供的小鼠BLC基因序列設(shè)計克隆擴增BLC的cDNA基因序列的引物,利用RT-PCR技術(shù)獲得小鼠BLC cDNA基因,通過限制性內(nèi)切酶酶切和T4酶連接,將BLC基因cDNA序列插入伯氏瘧原蟲UIS3基因敲除質(zhì)粒中,獲得重組伯氏瘧原蟲BLC轉(zhuǎn)基因重組質(zhì)粒。重組質(zhì)粒含有息瘧定抗性基因和綠色熒光蛋白的報告基因,可在轉(zhuǎn)化子中表達。篩選重組質(zhì)粒并經(jīng)酶切和DNA序列測定證實正確性后,體外轉(zhuǎn)染真核細胞。經(jīng)瓊脂糖凝膠電泳發(fā)現(xiàn)擴增的小鼠BLC基因cDNA序列分子量大小和預(yù)期值相一致,重組質(zhì)粒酶切結(jié)果和預(yù)期值相符,DNA序列測定顯示重組質(zhì)粒的目的基因閱讀框架準確無誤。從mRNA蛋白質(zhì)表達二個層面證明重組質(zhì)�？梢栽谡婧思毎杏行П磉_BLC基因。證明已成功構(gòu)建了伯氏瘧原蟲BLC轉(zhuǎn)基因重組質(zhì)粒(UIS3-/BLC+)。 伯氏瘧原蟲在轉(zhuǎn)化前經(jīng)體外短期培養(yǎng),利用密度梯度介質(zhì)Renografin分離子孢子用于電穿孔轉(zhuǎn)化。將構(gòu)建的伯氏瘧原蟲BLC轉(zhuǎn)基因重組質(zhì)粒經(jīng)酶切線性化后轉(zhuǎn)染伯氏瘧原蟲,獲得UIS3-/BLC+瘧原蟲株。將此瘧原蟲株經(jīng)尾靜脈注入小鼠體內(nèi),用息瘧定腹腔內(nèi)注射進行藥物篩選,經(jīng)2輪篩選后,得到了陽性的伯氏瘧原蟲轉(zhuǎn)化子。在熒光顯微鏡下觀察到經(jīng)息瘧定篩選的原蟲呈現(xiàn)綠色熒光；經(jīng)PCR檢測到了重組質(zhì)粒的存在,說明重組質(zhì)粒已正確的整合在伯氏瘧原蟲基因之中。 本研究進一步在小鼠伯氏瘧疾模型中,采用分子生物學(xué)、免疫學(xué)和組織學(xué)等技術(shù)對獲得的UIS3-/BLC+瘧原蟲株進行抗瘧機理研究。結(jié)果發(fā)現(xiàn)UIS3-/BLC+瘧原蟲株可以在肝細胞期有效生長,但未能有效發(fā)育轉(zhuǎn)化為紅內(nèi)期,具備了基因敲除減毒的特性。與UIS3子孢子免疫相比,我們發(fā)現(xiàn),UIS3-/BLC+子孢子免疫的小鼠可以有效產(chǎn)生體液和細胞免疫反應(yīng),抗體的水平和持續(xù)時間均明顯強于UIS3-子孢子,T淋巴細胞特異殺傷瘧原蟲的能力也明顯增強。體內(nèi)外抗瘧疾感染試驗結(jié)果也表明,UIS3-/BLC+子孢子作為疫苗較UIS3-具有更好的抑制瘧原蟲生長和轉(zhuǎn)化的作用。 本研究結(jié)果表明,基因工程敲除并轉(zhuǎn)入免疫增強基因的UIS3-/BLC+瘧原蟲子孢子是一種很好的瘧疾疫苗模式,具有較好的臨床應(yīng)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.
【學(xué)位授予單位】：海南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

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