【作者】 陈川；

【导师】 孙晗笑；

【作者基本信息】 暨南大学 ， 微生物与生化药学， 2010， 硕士

【摘要】 前期工作背景：在2004年,对恒河猴的cDNA文库进行筛选得到TRIM5α,一种能够抑制HIV-1感染的细胞质因子。恒河猴和短尾猴的TRIM5α能够抑制HIV-1感染而不能抑制SIVmac,与此相反,人的TRIM5α对上述病毒几乎没有什么抑制作用,具有能力抑制Y逆转录病毒N-MLV。然而,当有个别氨基酸改变时,人源的TRIM5α将具有和恒河猴TRIM5α相似水平的抗HIV-1功能。由于人抗异种动物的免疫应答反应的存在,异种蛋白在人体内往往被快速清除,其半衰期也较短,因此本实验室选择人源的TRIM5α(改构体)进行研究。以往关于TRIM5α的绝大多数研究,主要围绕在基因水平上进行的,例如,通过脂质体、病毒载体等结构将TRIM5α基因转运进入细胞使之大量表达,从而改变细胞的表型。在实际操作过程中,往往存在可操作性差,转运效率低,稳定性不好,生物毒性作用的缺点。所以本实验室前期已经构建出PTD-TRIM5α和TRIM5αH (R328-332)的大肠杆菌表达系统,但是其表达量不高。本实验室对PTD-TRIM5α和PTD-TRIM5αH(R328-332)的穿膜效率、对靶细胞的细胞毒性和抗HIV-1的能力进行了研究,表明PTD-TRIM5α和PTD-TRIM5αH(R328-332)在穿膜肽的作用下,能够进入细胞发挥生物学作用,对靶细胞也无毒性。虽然PTD-TRIM5α和PTD-TRIM5αH(R328-332)是人源的,但是本实验室进行了几个氨基酸的置换,所以其安全性有必要进一步考察,尤其是对正常人体细胞的毒性作用。到目前为止,TRIM5α限制HIV-1的机制已越来越清楚,但是,人们对于TRIM5α限制HIV-1复制的作用机制尚不完全清楚,许多问题还没有解决,大多停留在推测的水平。本实验室已经得出它直接与HIV-1的核衣壳蛋白gag结合,但它是否会导致核衣壳的重新定位、修饰或降解?[1]TRIM5α是否包含有一个泛素连接酶的亚单位,或者一个相似作用的SUMO (small ubiquitin-related modifier)转移酶的亚基?逆转录过程本身被TRIM5α阻断,还是DNA合成后降解的结果?TRIM5α还可能与其他什么蛋白结合?研究目的：1.将课题组前期构建的重组质粒pET28a转化大肠菌株BL21(DE3)α从基因水平上和蛋白质水平上,确定重组基因PTD-TRIM5α和PTD-TRIM5αH(R328-332)得到表达。2.通过对比本实验室构建的人TRIM5α嵌合体重组质粒在各种不同表达条件下的表达量,从而探索出目的蛋白在大肠杆菌中的最佳表达条件。3.天然人源的TRIM5α抗HIV-1的能力比较低,当突变的TRIM5αH(R328-332)[即I→M(328)、G→Q(330)、R→P(332)]基因用逆转录病毒载体转染细胞后,具有较好的抑制HIV-1的作用,本实验室进行了上述突变,所以其安全性有必要进一步考察。对靶细胞的细胞毒性,本研究进一步研究其对人体正常细胞的毒性。4.本实验室已经得出它直接与HIV-1的核衣壳蛋白gag结合,为深入了解TRIM5α的抗HIV-1的机制,运用共聚焦显微镜观察TRIM5α作用的亚细胞部位。5.确定TRIM5α的泛素化与其抗HIV-1的能力的关系。研究方法：1.在基因水平上应用酶切鉴定、PCR鉴定、基因测序,在蛋白水平上利用SDS-PAGE、Western-blotting、肽图指纹图谱鉴定,进行重组基因PTD-TRIM5α和PTD-TRIM5αH (R328-332)表达的鉴定。2.运用SDS-PAGE电泳分析重组蛋白在不同温度、IPTG的浓度、诱导时长、诱导时期与培养基的表达量,分别找出各个因素的目的蛋白表达量最大的最适条件。3.应用台盼蓝排斥实验和SunBioTMAm-Blue检测比色法检测PTD-TRIM5α和PTD-TRIM5αH(R328-332)的细胞毒性和对细胞增殖的影响。4.重组蛋白和细胞核分别用FITC和Hoechst荧光标记,在共聚焦显微镜观察下观察荧光的分布情况,从而确定重组蛋白作用的亚细胞结构部位。5.采用ELISA方法检测TRIM5的泛素化与其抗HIV-1的能力的关系。实验结果：1.在基因水平上和蛋白质水平上,都出现了目的条带或目标峰,确定重组基因PTD-TRIM5α和PTD-TRIM5αH (R328-332)得到表达。2.通过分析比较,重组质粒在30℃、IPTG的诱导浓度为0.5mmol/L、诱导时长为8h、菌液的OD值为0.6以及在TB培养基上培养人TRIM5α嵌合体蛋白表达量达到最大。3.在细胞样品中加入重组蛋白,3T3细胞的存活率都在83%以上,所以重组蛋白是安全无毒性的。4.分析荧光染料的颜色可知,重组蛋白只限定在细胞质内,没有进入细胞核内。5.当重组蛋白PTD-TRIM5α和PTD-TRIM5αH(R328-332)的浓度分别为100μg·ml-1、10μg-mL-1、1μg-mL-1、0.1μg-1、0.01μg-mL-1时,UBPL浓度的浓度分别为225.7pg·mL-1、160pg·mL-1、125.7 pg·mL-1、57.1 pg·mL-1、41.4 pg-mL"1和720 pg·mL-1、304.3 pg·mL-1、164.3 pg·mL-1、88.6 pg·mL-1、51.4 pg·mL-1。结论：1.在本实验室人员地努力下,成功地构建了表达重组蛋白PTD-TRIM5α和PTD-TRIM5αH(R328-332)的大肠杆菌原核表达系统,并且本研究在基因水平和蛋白质水平上得到鉴定。2.对重组蛋白PTD-TRIM5α和PTD-TRIM5αH(R328-332)的大肠杆菌原核表达系统的表达条件进行了优化,使得重组蛋白质得率有显著地提高。3.之前本实验室已经证明了重组蛋白PTD-TRIM5α和PTD-TRIM5αH(R328-332)对靶细胞无毒性,本研究进一步证明了重组蛋白对人体正常细胞是安全无毒性作用。4.通过荧光标记,在共聚焦显微镜下观察重组蛋白PTD-TRIM5α和PTD-TRIM5αH (R328-332)的抗HIV-1的作用限定在细胞质内,不进入细胞核,重组蛋白没有进入细胞核直接抑制HIV-1的复制,故可确定重组蛋白是通过阻碍HIV-1复制前的或复制后的生命活动过程,从而达到抗HIV-1的作用。5.通过ELISA法检测发现,重组蛋白PTD-TRIM5α和PTD-TRIM5αH (R328-332)的泛素化作用与抗HIV-1能力呈一定地线性关系。由此可知,重组蛋白有可能是通过促进对特异性病毒蛋白质的水解,达到抗病毒作用。更多还原

【Abstract】 Study BackgroundIn 2004, TRIM5a was screened from rhesus monkeys, which is a cytoplasm factor can inhibit to the HIV-1, Rhesus monkeys TRIM5a and that of macaques can inhibit to HIV-1, but not SIVmac, on the contrary, human TRIM5a can’t inhibit to those viruses, has the ability of inhibition to the Y-retrovirus N-MLV. However, when several amino acids change, the human TRIM5 alpha will have similar level of anti-HIV-1 as the Rhesus TRIM5a. In addition, because of the immune response of human has anti-Heterogeneous animal, heterologous protein in the human body is often quick to be removed, and the half-life is shorter, so human TRIM5a(mutant) was chosen for studying in our laboratory.But in the past, the majority of the researches about TRIM5a were finished by gene transfection, For example, TRIM5a gene was conveyed into the cells by liposome or viral vector, but there are many problems during the operative procedure, such as poor feasibility, low efficiency and stability, low transfection rate, biological toxicity, so the E.coil expression systems of PTD-TRIM5a and TRIM5aH (R328-332) were constructed early in our lab, but the efficiency of it were not high.The transmembrane efficientcy, cytotoxicity to target cells and inhibition to HIV-1 of PTD-TRIM5a and TRIM5aH (R328-332) were researched before, shows that PTD-TRIM5a and TRIM5aH (R328-332) can entry into cells, can play biological effect on target cells and have no toxicity. Although PTD-TRIM5a and TRIM5aH (R328-332) are from human, several amino acids were replaced by our lab, the safety of it is necessary to study for next, especially for normal human cells.So far, the mechanism of TRIM5a inhibition to HIV-1 has become more and more clear, however, the reaction of TRIM5a restricted HIV-1 replication is not yet entirely clear, many questions need to be resolved, most remain at the level of speculation, In our lab, TRIM5a can bind with gag of HIV-1 capsid protein directly has studied, but it will result in reposition, modification or degradation of nucleocapsid also need to research. Whether TRIM5a contains a ubiquitin ligase subunit, or a subunit which is similar with small ubiquitin-related modifier, the process of reverse is blocked by TRIM5a, or synthesized DNA were degraded, or other proteins were combined with TRIM5a, all of this will remain to study.Objective:1. The recombinant plasmid of pET28a previously constructed was transformed into colorectal strain BL21 (DE3), testing PTD-TRIM5a and TRIM5aH (R328-332) were expressed on level of gene and protein.2. To optimize the expression of human TRIM5a chimera protein in E. coli DE3, we have compared with expression product of human TRIM5a Recombinant Plasmid which was constructed in our lab in different conditions.3. Although PTD-TRIM5a and TRIM5aH (R328-332) are from human, several amino acids were replaced by our lab, the safety of it is necessary to study for next, cytotoxicity of normal human cells were researched in this study.4. In our lab, TRIM5a can bind with gag of HIV-1 capsid protein directly has studied, in order to study the mechanism of TRIM5a inhibition to HIV-1, Subcellular localization of TRIM5a inhibition to HIV-1 was analyzed by confocal laser screening microscopy.5. To be defined the relationship of ubiquitination and capacity of TRIM5a.Methods1. Using enzyme digestion, PCR, gene sequencing on the gene level, and SDS-PAGE, Western-blotting, peptide mass fingerprint (PMF) analysis, to test the expression of PTD-TRIM5a and PTD-TRIM5aH (R328-332).2. Protein products which expressed in different temperature, concentration of IPTG, duration of induce, induce time, medium were analyzed by SDS-PAGE, maximal expression of each condition was found out separately.3. Cytotoxicity of PTD-TRIM5a and PTD-TRIM5aH (R328-332) to normal human cells was studied by trypanblau and SunBioTMAm-Blue.4. Recombinant protein and nucleus were labeled with FITC and Hoechst separately, the distribution of fluorescence was observed under confocal laser screening microscope, Subcellular localization of TRIM5a inhibition to HIV-1 was analyzed.5. To be defined the relationship of ubiquitination and capacity of TRIM5a by ELISA.Results1. Target bands or peaks were appeared on level of gene and protein, so PTD-TRIM5a and PTD-TRIM5aH (R328-332) had expressed.2. Through analysis and comparison, maximal expression of recombinant plasmid was induced by 0.5mmol/L IPTG at 30℃for 8 h when OD value of bacterium was 0.6.3. Recombinant protein was added into sample,83% 3T3 cells were survived, so the recombinant protein was safe.4. Analysis of the color of fluorescent, shows that the recombinant protein is only limited into the cytoplasm, not enter nucleus.5. when concentration of PTD-TRIM5a and PTD-TRIM5aH(R328-332) were 100μg-mL-110μg-mL-1 1μgmL-1、0.1μg-mL-1、0.01μg-mL"1 respectively, its’ concentration of UBPL were about 225.7pg-mL-1、160 pg-mL-1、125.7 pg-mL-1、57.1 pg-mL、41.4 pg-mL-1 and 720 pg-mL-1、304.3 pg-mL-1、164.3 pg-mL-1、88.6 pg-mL-1、51.4 pg-mL-1 separately.Conclusions1. As effort of our lab, prokaryotic expression systems of E.coli of PTD-TRIM5a and PTD-TRIM5aH(R328-332) were successfully constructed, it was identified on gene and protein level.2. After the optimization of individual condition factor, recombinant protein expression level significantly enhanced after the optimal conditions.3. Although PTD-TRIM5a and TRIM5aH (R328-332) are from human, several amino acids were replaced by our lab, the safety of it is necessary to study for next, cytotoxicity of normal human cells were researched, and it is safe.4. Analysis of the color of fluorescent, shows that the recombinant protein is only limited into the cytoplasm, not enter nucleus, so recombinant protein can not entry into nucleus for inhibiting to replication of HIV-1.5. From the test of ELISA, a certain linear relationship was showed between ubiquitination of recombinant protein and ability of anti-HIV-1 of it.更多还原