NT1基因敲除的减毒伯氏疟原虫构建及其抗肿瘤作用研究

发布时间：2018-05-01 15:01

本文选题：伯氏疟原虫 + NT1基因　；参考：《安徽大学》2017年硕士论文

【摘要】：疟疾是世界三大传染病之一,以按蚊为传播媒介,致病病原体是疟原虫。目前,32亿人处在疟疾威胁之下,其中95%的疟疾死亡是由于恶性疟原虫感染导致的。肺癌是全球最常见的恶性肿瘤之一,发病率和死亡率都位居癌症新增发病和死亡率之首,传统的手术、化疗、放疗及其联合疗法由于各自的局限性在控制肺癌的发展已经遇到很大的瓶颈,例如缺乏特异性,生物疗法越来越受到重视。本实验室前期研究发现,疟原虫通过广泛激活机体的天然免疫系统和诱导一定的针对肿瘤的特异性抗原,从而显著地抑制小鼠Lewis肺癌的生长和延长荷瘤小鼠的生存期。但是疟原虫具有一定的毒副作用,我们拟对疟原虫进行减毒,提高疟原虫治疗肺癌的安全性。疟原虫本身没有嘌呤的合成途径,只能从宿主中获得嘌呤碱或者嘌呤核苷,依赖补救合成途径满足疟原虫的裂体增殖需求。核苷转运蛋白1(NT1)是疟原虫合成嘌呤途径的一个关键酶,负责将嘌呤核苷和嘌呤碱基转运到疟原虫的膜内。在C57BL/6小鼠中Plasmodium berghei ANKA(P.b ANKA)是致死性的鼠疟原虫株,Pb.ANKA感染C57BL/6小鼠是研究减毒疟原虫的最佳模型。本研究首先构建双交换同源重组的质粒载体pL0018-NT1,质粒上含有GFP标记基因和tgdhfr/ts抗性基因,使用电转化方式将质粒转进P.b ANKA疟原虫中,经过乙胺嘧啶药筛,第一次单克隆化得到GFP表达和位点特异性整合的2个克隆8#和9#,其中8#克隆经过第二次单克隆化,得到稳定NT1基因敲除和位点特异性整合的P.b ANKA-ΔNT1-8C1克隆。观察发现野生型P.b ANKA感染小鼠后小鼠很快全部死亡,NT1敲除的P.b ANKA-ΔNT1虫株在接种C57BL/6小鼠后,能形成高达85%的感染率的原虫血症,感染周期能达到80天,6只感染的小鼠有5只不死亡,总体生存期显著延长。在P.b ANKA-Δ NT1感染小鼠38天时间里,在100倍油镜下观察发现配子体数显著降低,每100个白细胞最高只能观察到48个配子体,在接种2～18天内,配子体数在0～25个数量范围内波动,远低于野生型最多时400个配子体的水平。疟原虫的配子体数目的降低有利于阻断疟原虫在按蚊体内的有性生殖,是药物减毒和辐照减毒策略所不具有的安全性优势,避免疟原虫通过按蚊在人群中大量传播。我们之前研究发现疟原虫感染荷瘤小鼠时间越长,抑制肿瘤效果越好。P.b ANKA-ΔNT1减毒虫株红内期感染长达80天,比约氏疟原虫30天要更长。P.b ANKA-ΔNT1比野生型P.b ANKA感染Lewis肺癌皮下接种C57BL/6小鼠更加显著的抑制肿瘤生长的效果,中位生存期也要延长五天。NT1基因敲除的P.b ANKA-ΔNT1虫株显著降低疟原虫对小鼠的毒副作用,从致死型虫株转变为非致死性虫株,具有更长的感染周期,同时显著降低外周血中的配子体,提供了更加有效和安全的减毒疟原虫,可以作为肺癌疫苗的肿瘤抗原表达载体,为肿瘤的生物免疫治疗提供新的方法和思路,同时还可以作为减毒全虫活疫苗,用于保护人类健康和消灭疟疾。
[Abstract]:Malaria is one of the three major infectious diseases in the world. The Anopheles mosquito is the medium and the pathogenic pathogen is the Plasmodium. At present, 3 billion 200 million people are under the threat of malaria, of which 95% of the deaths are caused by the infection of Plasmodium falciparum. Lung cancer is one of the most common malignant tumors in the world. The incidence and mortality of the disease are all in the new disease and death of cancer. In the first place, traditional surgery, chemotherapy, radiotherapy and combined therapy have encountered great bottlenecks in controlling the development of lung cancer. For example, the lack of specificity, biologic therapy is becoming more and more important. The specific antigen of the tumor significantly inhibits the growth of Lewis lung cancer in mice and prolongs the survival period of the tumor bearing mice. However, the Plasmodium has a certain toxic side effect. We intend to reduce the toxicity of the Plasmodium to improve the safety of the Plasmodium in the treatment of lung cancer. The parasite itself has no synthetic route of the MTX, only the purine base is obtained from the host. Or purine nucleoside, dependent on the remedial synthesis pathway to meet the proliferation demand of the Plasmodium, a key enzyme for the Plasmodium purine pathway, which is responsible for transshipment of the purine nucleosides and purine bases into the Plasmodium Plasmodium membrane. In C57BL/6 mice, Plasmodium berghei ANKA (P.b ANKA) is fatal to the Plasmodium Plasmodium strain. Pb.ANKA infected C57BL/6 mice is the best model for the study of Plasmodium Plasmodium. First, we construct a double exchange homologous recombinant plasmid vector pL0018-NT1, which contains GFP marker gene and tgdhfr/ts resistance gene. The plasmid is transferred into the Plasmodium P.b ANKA by electric transformation. The first monoclonal antibody is obtained by ethamidine sieving. GFP expression and site specific integration of 2 clones 8# and 9#, in which 8# clones were clones of P.b ANKA- Delta NT1-8C1 that stabilized NT1 gene knockout and site specific integration. After that, the infection rate of protozoa could be up to 85%, the infection cycle could reach 80 days, 5 of the 6 infected mice were not dead, and the overall survival time was prolonged significantly. In the 38 days of P.b ANKA- Delta NT1 infected mice, the number of gametes decreased significantly under 100 times of oil microscope, and only 48 gametophytes could be observed at the highest level of every 100 white cells. Within 2~18 days, the number of gametes fluctuates in 0~25 quantities, far below the level of 400 gametophytes at the maximum of the wild type. The reduction of the number of gametophytes of the Plasmodium is beneficial to blocking the sexual reproduction of the Plasmodium in the Anopheles mosquitoes. It is the safety advantage that the drug reduction and radiation reduction strategies do not have, to avoid the passing of malaria parasites by the malaria parasite. The longer the mosquitoes spread in the population, the longer we found that the longer the infected mice were infected with Plasmodium, the better the effect of the tumor suppressing.P.b ANKA- Delta NT1 in the erythroid infection for up to 80 days, and the longer.P.b ANKA- Delta NT1 than Plasmodium komoya, which was more significant than that of the wild type P.b ANKA infected Lewis lung cancer subcutaneous inoculation C57BL/6 mice. The effect of the growth of the tumor, the median survival period also extended the five days of.NT1 gene knockout P.b ANKA- Delta NT1 to significantly reduce the toxic and side effects of the Plasmodium to mice, from the death type to the non lethal strain, with a longer period of infection, and a significant reduction in the gametophyte in the peripheral blood, providing a more effective and safe reduction of the toxicity. Plasmodium, which can be used as a tumor antigen expression vector for lung cancer vaccine, provides new methods and ideas for the biological immunotherapy of cancer, and can also be used as a live attenuated live vaccine to protect human health and eliminate malaria.

【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-36

【参考文献】