特异性siRNA在肺结核疾病核酸疫苗开发中的研究
发布时间：2019-02-21 11:45
【摘要】： 结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)所导致,以呼吸系统感染为主的慢性传染病。卡介苗(BCG)是目前唯一的预防TB的疫苗,但其保护效果不稳定(保护率为0-80%),由于MTB耐药株的流行与HIV的合并感染以及人口流动的增加等因素,进一步加剧了MTB对人类的威胁,因此迫切需要研制安全新型、更加有效的TB疫苗。 由于MTB感染人体后,主要被巨噬细胞吞噬,未被机体免疫系统清除而潜伏下来的结核分枝杆菌也主要寄生于巨噬细胞内,而巨噬细胞发生凋亡后可杀死寄生于其内的结核分枝杆菌。因此,巨噬细胞的凋亡情况对于寄生于其中的结核分枝杆菌的命运至关重要。而巨噬细胞中较高表达抑制巨噬细胞凋亡的Mcl-1L蛋白,因此,通过抑制Mcl-1L蛋白表达来促使巨噬细胞凋亡成为治疗肺结核疾病的可能。 因此,本研究首先采用siRNA技术抑制负调因子Mcl-1L的表达,针对mcl-1构建两组siRNA表达质粒psimcl-1-1和psimcl-1-2,即将编码siRNA的目的双链DNA克隆到siRNA表达载体psiRNA-hH1neo质粒上,将其转化到感受态细胞GT116,经过蓝白筛选、酶切和测序等鉴定克隆的正确性;然后两组重组质粒经过纯化后转染相关细胞,采用实时荧光定量PCR方法鉴定siRNA对mcl-1 mRNA的抑制情况以及应用Western Bolt方法鉴定细胞中Mcl-1蛋白表达情况,通过比较,筛选出具抑制效果较好siRNAmcl-1-2片段;最后将筛选的特异性siRNA和融合抗原基因共同构建到具更高转导效率的AAV载体上,联合双因素作用来增强疫苗的保护效力,尝试开发结核病新型核酸疫苗。这种方法的成功将为其它传染病的预防和治疗提供思路和手段。
[Abstract]:Tuberculosis (Tuberculosis,TB) is a chronic infectious disease caused by mycobacterium tuberculosis (Mycobacterium tuberculosis,MTB). BCG (BCG) is the only vaccine to prevent TB at present, but its protective effect is unstable (protection rate is 0-80%). Due to the co-infection of HIV and the prevalence of MTB resistant strains, and the increase of population mobility, etc. The threat of MTB to human beings is further aggravated, so it is urgent to develop a new safe and effective TB vaccine. After MTB infection, it was mainly swallowed by macrophages, and mycobacterium tuberculosis, which was not cleared by the body's immune system, was mainly parasitic in macrophages. However, macrophage apoptosis can kill Mycobacterium tuberculosis parasitic in it. Therefore, the apoptosis of macrophages is critical to the fate of Mycobacterium tuberculosis. However, the higher expression of Mcl-1L protein in macrophages inhibits the apoptosis of macrophages. Therefore, it is possible to treat pulmonary tuberculosis by inhibiting the expression of Mcl-1L protein to induce the apoptosis of macrophages. Therefore, siRNA technique was used to inhibit the expression of negative modulation factor Mcl-1L, and two groups of siRNA expression plasmids psimcl-1-1 and psimcl-1-2, were constructed for mcl-1. The target double-stranded DNA encoding siRNA was cloned into the psiRNA-hH1neo plasmid of siRNA expression vector, and transformed into the competent cell GT116, after blue-white screening, restriction endonuclease digestion and sequencing to identify the correctness of the clone. Then the two groups of recombinant plasmids were purified and transfected into related cells. The inhibition of mcl-1 mRNA by siRNA and the expression of Mcl-1 protein in cells were identified by real-time fluorescence quantitative PCR and Western Bolt. Screening siRNAmcl-1-2 fragments with good inhibitory effect; Finally, the screened specific siRNA and fusion antigen gene were constructed into the AAV vector with higher transduction efficiency, combined with double factors to enhance the protective effect of the vaccine, and to try to develop a new nucleic acid vaccine for tuberculosis. The success of this method will provide ideas and means for the prevention and treatment of other infectious diseases.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392;Q789
本文编号：2427467
[Abstract]:Tuberculosis (Tuberculosis,TB) is a chronic infectious disease caused by mycobacterium tuberculosis (Mycobacterium tuberculosis,MTB). BCG (BCG) is the only vaccine to prevent TB at present, but its protective effect is unstable (protection rate is 0-80%). Due to the co-infection of HIV and the prevalence of MTB resistant strains, and the increase of population mobility, etc. The threat of MTB to human beings is further aggravated, so it is urgent to develop a new safe and effective TB vaccine. After MTB infection, it was mainly swallowed by macrophages, and mycobacterium tuberculosis, which was not cleared by the body's immune system, was mainly parasitic in macrophages. However, macrophage apoptosis can kill Mycobacterium tuberculosis parasitic in it. Therefore, the apoptosis of macrophages is critical to the fate of Mycobacterium tuberculosis. However, the higher expression of Mcl-1L protein in macrophages inhibits the apoptosis of macrophages. Therefore, it is possible to treat pulmonary tuberculosis by inhibiting the expression of Mcl-1L protein to induce the apoptosis of macrophages. Therefore, siRNA technique was used to inhibit the expression of negative modulation factor Mcl-1L, and two groups of siRNA expression plasmids psimcl-1-1 and psimcl-1-2, were constructed for mcl-1. The target double-stranded DNA encoding siRNA was cloned into the psiRNA-hH1neo plasmid of siRNA expression vector, and transformed into the competent cell GT116, after blue-white screening, restriction endonuclease digestion and sequencing to identify the correctness of the clone. Then the two groups of recombinant plasmids were purified and transfected into related cells. The inhibition of mcl-1 mRNA by siRNA and the expression of Mcl-1 protein in cells were identified by real-time fluorescence quantitative PCR and Western Bolt. Screening siRNAmcl-1-2 fragments with good inhibitory effect; Finally, the screened specific siRNA and fusion antigen gene were constructed into the AAV vector with higher transduction efficiency, combined with double factors to enhance the protective effect of the vaccine, and to try to develop a new nucleic acid vaccine for tuberculosis. The success of this method will provide ideas and means for the prevention and treatment of other infectious diseases.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392;Q789
【引证文献】
相关硕士学位论文 前1条
1 刘佳;中枢神经系统感染患者血尿酸水平及IL-10和TNF-α在人PBMCs体外感染BCG中的研究[D];中山大学;2012年
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