抗结核基因疫苗及基因重组卡介苗的免疫效应研究

发布时间：2018-06-06 14:04

本文选题：结核分枝杆菌 + 基因疫苗　；参考：《四川大学》2007年博士论文

【摘要】： 结核病是人类主要的传染性疾病之一。全球每年新发结核病人数超过800万，死亡人数约300万，全世界1／3人口受到结核菌感染。近年来HIV的广泛流行及多重耐药结核菌株的出现使结核病的发病率和死亡率呈上升趋势。我国结核病疫情在全球属高流行地区，结核病人数居全世界第二位，是全球22个结核病高负担国家之一。据国家卫生部2006年3月24日的报告，结核病的发病率和死亡率都居于传染病首位。世界卫生组织早在1993年宣布“全球结核病紧急状态”，并指出应优先发展有效的结核病疫苗。目前用于预防结核病的唯一疫苗是卡介苗(BCG)，它是牛分枝杆菌的减毒活菌苗株。由于反复传代的原因，卡介苗一些重要的免疫保护性抗原基因已发生变异，免疫保护效果也大不如前。免疫保护力在不同人群中差异极大，为0-80％；通过连续免疫接种BCG企图降低肺结核发生率的实验结果也令人失望，因此，改造BCG、开发新疫苗已成为研究热点。目前结核病疫苗主要有：基因工程重组BCG、亚单位疫苗、基因疫苗、营养缺陷型疫苗等类型。白细胞介素12(IL-12)是由单核细胞、巨噬细胞分泌产生的一种细胞因子，具有广泛的生物学活性，是连接天然免疫和获得性免疫的功能性桥梁，是目前发现对体内免疫活性细胞诱导和调节作用最强、范围最广的细胞因子；在诱导Th0细胞向Th1型细胞发育过程中发挥重要作用，可明显增强细胞免疫功能。本研究报道了IL-12真核表达质粒及IL-12基因重组卡介苗的构建，并对IL-12与结核杆菌免疫保护性抗原基因疫苗联合免疫及IL-12基因重组卡介苗的免疫原性以及在调节机体免疫应答方面的作用进行了研究。IL-12在正常情况下为非组成性表达，我们首先经体外分别诱导人白血病细胞株THP-1和HL-60，得到IL-12 p40、p35及p40-p35融合基因。将IL-12基因克隆入真核表达质粒pcDNA3.1(+)／myc-His中，获得IL-12基因真核表达质粒pcDNA-IL-12，并将其转染COS-7细胞，通过RT-PCR及ELISA检测进行了鉴定。ELISA检测证实转染后的COS-7细胞能分泌表达IL-12。为研究细胞因子和结核菌保护性抗原基因疫苗共同免疫后，对小鼠体内免疫应答的诱导及小鼠免疫功能的影响，我们将IL-12和ESAT-6抗原真核表达质粒共同肌肉免疫BALB／c小鼠，结果发现IL-12与ESAT-6基因联合免疫，较单独的ESAT-6基因免疫，刺激机体产生更加强烈而稳定的细胞免疫应答。IL-12与ESAT-6联合免疫，ESAT-6使用剂量是ESAT-6单独免疫时的一半，但是诱导的免疫反应却明显强于ESAT-6单独免疫，而与BCG免疫相当。另外，我们将IL-12基因连接到pMV361载体上，构建含人IL-12基因的重组大肠杆菌—分枝杆菌穿梭质粒rpMV-12，，通过电穿孔的方法转化BCG。该基因重组BCG经卡那霉素抗性筛选和PCR扩增鉴定，进一步通过重组BCG诱导表达后的SDS-PAGE电泳检测，筛选得到能分泌表达IL-12基因的重组BCG菌株rBCG-12。接着我们用IL-12基因重组BCG(rBCG-12)和传统BGG分别免疫小鼠，观察免疫后不同时间点小鼠体内细胞免疫和体液免疫的变化。将rBCG-12和BCG皮下接种BALB／c小鼠(10~6 CFU)，分别在免疫接种后第6、8、10、12周时，测定血清特异性抗体水平，脾淋巴细胞增殖能力，IFN-γ和IL-4分泌量。实验结果显示，rBCG-12免疫组的脾淋巴细胞增殖能力、IFN-γ分泌水平均明显高于对照组和BCG免疫组，并且维持的时间也更长。而rBCG-12免疫组、BCG免疫组和对照组血清特异性抗体水平和IL-4分泌水平差异没有统计学意义。
[Abstract]:Tuberculosis is one of the major infectious diseases in the world . The number of new TB patients worldwide is over 8 million , the number of deaths is about 3 million , and 1 / 3 of the world ' s population is affected by TB . In recent years , the prevalence of tuberculosis and the emergence of multiple - resistant TB strains have caused tuberculosis infection . In recent years , the incidence and mortality of tuberculosis have been the first in the world . According to the report of the Ministry of Health of the Ministry of Health on March 24 , 2006 , the World Health Organization announced the Global Tuberculosis Emergency in 1993 , and noted that effective tuberculosis vaccines should be prioritized .

The only vaccine used to prevent tuberculosis is BCG , which is the attenuated live vaccine strain of Mycobacterium species . Because of the repeated passage , some important immune protective antigen genes of BCG have been mutated , and the immune protection effect is too high . The difference of immune protection force among different populations is very large , which is 0 - 80 % ;
The results of BCG and development of new vaccines have become the focus of research . At present , tuberculosis vaccine is mainly composed of recombinant BCG , subunit vaccine , gene vaccine and nutrition deficient vaccine .

Interleukin - 12 ( IL - 12 ) is a cytokine produced by monocytes and macrophages . It has a wide range of biological activities . It is a functional bridge linking innate immunity and acquired immunity . It is the most widely used cytokine in the induction and regulation of immune active cells in vivo .
It plays an important role in the development of Th1 - type cells by inducing Th0 cells , which can obviously enhance cellular immunity .

IL - 12 was cloned into eukaryotic expression plasmid pcDNA3.1 ( + ) / myc - His to obtain IL - 12 p40 , p35 and p40 - p35 fusion gene . The IL - 12 gene was cloned into eukaryotic expression plasmid pcDNA3.1 ( + ) / myc - His .

IL - 12 and ESAT - 6 were immunized with ESAT - 6 gene . The results showed that IL - 12 and ESAT - 6 were immunized with ESAT - 6 gene . The results showed that IL - 12 and ESAT - 6 were immunized with ESAT - 6 gene and stimulated the organism to produce stronger and stable cellular immune response . The dose of ESAT - 6 was half that of ESAT - 6 alone , but the induced immune response was stronger than that of ESAT - 6 alone , but it was comparable to BCG vaccination .

In addition , the recombinant BCG strain rBCG - 12 capable of secreting IL - 12 gene was further screened by the recombinant BCG - induced SDS - PAGE electrophoresis . The recombinant BCG strain rBCG - 12 expressing IL - 12 gene was further screened by recombinant BCG - induced SDS - PAGE electrophoresis .

rBCG - 12 and BCG were subcutaneously injected into BALB / c mice ( 10 - 6 CFU ) . The levels of serum - specific antibody , spleen lymphocyte proliferation , IFN - 纬 and IL - 4 secretion were measured at 6 , 8 , 10 and 12 weeks after immunization respectively .
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【引证文献】