特異性siRNA在肺結(jié)核疾病核酸疫苗開發(fā)中的研究
發(fā)布時間：2019-02-21 11:45
【摘要】： 結(jié)核病(Tuberculosis,TB)是由結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)所導(dǎo)致,以呼吸系統(tǒng)感染為主的慢性傳染病�？ń槊�(BCG)是目前唯一的預(yù)防TB的疫苗,但其保護效果不穩(wěn)定(保護率為0-80%),由于MTB耐藥株的流行與HIV的合并感染以及人口流動的增加等因素,進一步加劇了MTB對人類的威脅,因此迫切需要研制安全新型、更加有效的TB疫苗。 由于MTB感染人體后,主要被巨噬細胞吞噬,未被機體免疫系統(tǒng)清除而潛伏下來的結(jié)核分枝桿菌也主要寄生于巨噬細胞內(nèi),而巨噬細胞發(fā)生凋亡后可殺死寄生于其內(nèi)的結(jié)核分枝桿菌。因此,巨噬細胞的凋亡情況對于寄生于其中的結(jié)核分枝桿菌的命運至關(guān)重要。而巨噬細胞中較高表達抑制巨噬細胞凋亡的Mcl-1L蛋白,因此,通過抑制Mcl-1L蛋白表達來促使巨噬細胞凋亡成為治療肺結(jié)核疾病的可能。 因此,本研究首先采用siRNA技術(shù)抑制負調(diào)因子Mcl-1L的表達,針對mcl-1構(gòu)建兩組siRNA表達質(zhì)粒psimcl-1-1和psimcl-1-2,即將編碼siRNA的目的雙鏈DNA克隆到siRNA表達載體psiRNA-hH1neo質(zhì)粒上,將其轉(zhuǎn)化到感受態(tài)細胞GT116,經(jīng)過藍白篩選、酶切和測序等鑒定克隆的正確性;然后兩組重組質(zhì)粒經(jīng)過純化后轉(zhuǎn)染相關(guān)細胞,采用實時熒光定量PCR方法鑒定siRNA對mcl-1 mRNA的抑制情況以及應(yīng)用Western Bolt方法鑒定細胞中Mcl-1蛋白表達情況,通過比較,篩選出具抑制效果較好siRNAmcl-1-2片段;最后將篩選的特異性siRNA和融合抗原基因共同構(gòu)建到具更高轉(zhuǎn)導(dǎo)效率的AAV載體上,聯(lián)合雙因素作用來增強疫苗的保護效力,嘗試開發(fā)結(jié)核病新型核酸疫苗。這種方法的成功將為其它傳染病的預(yù)防和治療提供思路和手段。
[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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392;Q789
本文編號：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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392;Q789
【引證文獻】
相關(guān)碩士學(xué)位論文 前1條
1 劉佳;中樞神經(jīng)系統(tǒng)感染患者血尿酸水平及IL-10和TNF-α在人PBMCs體外感染BCG中的研究[D];中山大學(xué);2012年
，本文編號：2427467
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