PTD-Trim5α和PTD-Trim5αH（R328-332）在大肠杆菌中的表达优化及其作用机制研究
发布时间：2018-08-23 08:46
【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。
[Abstract]:Background:
In 2004, the rhesus monkey cDNA library was screened for TRIM5a, a cytoplasmic factor that inhibits HIV-1 infection. The rhesus monkey and macaque TRIM5a inhibited HIV-1 infection but not SIVmac. On the contrary, human TRIM5a had little inhibition on the above viruses and had the ability to inhibit Y retrovirus N-MLV. However, when individual amino acids are changed, human TRIM5a will have the same level of anti-HIV-1 function as rhesus monkey TRIM5a. Because of the existence of human anti-xenogeneic immune response, xenogeneic proteins in the human body are often quickly cleared, and their half-lives are also shorter, so our laboratory selected human TRIM5a (modified) for research.
Most of the previous studies on TRIM5a have been carried out at the gene level. For example, TRIM5a gene is transported into cells by liposome, viral vectors and so on to express in large quantities, thus changing the cell phenotype. So we have constructed the expression system of PTD-TRIM5a and TRIM5alpha H (R328-332) in E. coli, but the expression level is not high.
The penetrating efficiency of PTD-TRIM5alpha and PTD-TRIM5alpha H (R328-332) and the cytotoxicity and anti-HIV-1 ability of target cells were studied in this laboratory. The results showed that PTD-TRIM5alpha and PTD-TRIM5alpha H (R328-332) could enter the cells to play biological roles and have no toxicity to target cells under the action of transmembrane peptides. - 332) Human, but the laboratory has carried out several amino acid substitutions, so its safety needs to be further investigated, especially the toxic effects on normal human cells.
Up to now, the mechanism of TRIM5a restricting HIV-1 has become more and more clear. However, the mechanism of TRIM5a restricting HIV-1 replication is still unclear. Many problems remain unsolved, mostly at the speculative level. RELOCATION, MODIFICATION OR DEGRADATION? [1] Does TRIM5a contain a ubiquitin ligase subunit, or a similar SUMO (small ubiquitin-related modifier) transferase subunit? Is the reverse transcription process itself blocked by TRIM5a, or is it the result of degradation after DNA synthesis? What other proteins may TRIM5a bind to?
Research purposes:
1. The recombinant plasmid pET28a was transformed into E. coli strain BL21 (DE3) alpha, and the recombinant genes PTD-TRIM5alpha and PTD-TRIM5alphaH (R328-332) were confirmed to be expressed at gene level and protein level.
2. By comparing the expression of recombinant human TRIM5a chimeric plasmid constructed in our laboratory under different expression conditions, the optimal expression conditions of the target protein in E.coli were explored.
3. Natural human TRIM5a has low anti-HIV-1 ability. When the mutant TRIM5alpha H (R328-332) [i.e. I M (328), G Q (330), R P (332)] gene is transfected into cells by retroviral vectors, it has a better anti-HIV-1 effect, so the safety of the mutant TRIM5alpha H (R328-332) gene should be further investigated. Toxicity, this study further studies its toxicity to human normal cells.
4. In order to understand the mechanism of TRIM5a's anti-HIV-1 action, the subcellular sites of TRIM5a were observed by confocal microscopy.
5. to determine the relationship between ubiquitination of TRIM5 and its ability to resist HIV-1.
Research methods:
1. The recombinant gene PTD-TRIM5a and PTD-TRIM5alpha H (R328-332) were identified by enzyme digestion, PCR, gene sequencing, SDS-PAGE, Western-blotting and peptide fingerprint analysis.
2. SDS-PAGE electrophoresis was used to analyze the expression of recombinant protein in different temperature, IPTG concentration, induction time, induction period and culture medium, and to find out the optimum conditions for the maximum expression of the target protein in each factor.
3. The cytotoxicity of PTD-TRIM5a and PTD-TRIM5alpha H (R328-332) and its effect on cell proliferation were detected by Trypan blue exclusion test and SunBioTMAm-Blue assay.
4. The recombinant protein and nucleus were labeled with FITC and Hoechst fluorescence respectively, and the distribution of fluorescence was observed under confocal microscope to determine the subcellular structure of the recombinant protein.
5. the relationship between the ubiquitination of TRIM5 and its ability to resist HIV-1 was detected by ELISA.
Experimental results:
1. Target bands or peaks appeared at both gene level and protein level, and the recombinant genes PTD-TRIM5alpha and PTD-TRIM5alphaH (R328-332) were confirmed to be expressed.
2. The results showed that the expression of TRIM5a chimeric protein reached the maximum at 30 C, the induction concentration of IPTG was 0.5mmol/L, the induction time was 8 h, the OD value of bacterial fluid was 0.6, and the expression level of TRIM5a chimeric protein in TB medium was the highest.
3. The survival rate of 3T3 cells was above 83% when the recombinant protein was added to the cell samples, so the recombinant protein was safe and nontoxic.
4. analyzing the color of fluorescent dyes, it is known that the recombinant protein is only restricted to the cytoplasm and does not enter the nucleus.
5.When the concentrations of recombinant protein PTD-TRIM 5 alpha and PTD-TRIM 5 alpha H (R328-332) and PTD-TRIM 5 alphaH (R328-332) were 100 \\\ ml-1,10 \\\\, 10 \\\\\\\\, 1, 1\\\\\\\\\\\\\\\\\\\\\\\\\\\\1,164.3 pg.mL-1,88.6 pg.mL-1,51.4 pg.mL-1.
Conclusion:
1. The prokaryotic expression system of recombinant protein PTD-TRIM5alpha and PTD-TRIM5alpha H (R328-332) was successfully constructed with the efforts of our lab staff, and this study was identified at the level of gene and protein.
2. The prokaryotic expression system of recombinant protein PTD-TRIM5a and PTD-TRIM5alphaH (R328-332) was optimized, and the yield of recombinant protein was significantly increased.
3. The recombinant protein PTD-TRIM5a and PTD-TRIM5alphaH (R328-332) have been proved to be non-toxic to target cells in our laboratory. This study further proves that the recombinant protein is safe and non-toxic to human normal cells.
4. The anti-HIV-1 effect of recombinant protein PTD-TRIM5alpha and PTD-TRIM5alpha H (R328-332) was observed under confocal microscope by fluorescence labeling. The recombinant protein did not enter the nucleus and inhibited the replication of HIV-1 directly. Therefore, it was determined that the recombinant protein inhibited the life of HIV-1 before or after replication by blocking the replication of HIV-1. Activity process, so as to achieve the anti HIV-1 effect.
5. ELISA showed that the ubiquitination of recombinant protein PTD-TRIM5a and PTD-TRIM5alpha H (R328-332) was linearly related to the ability of anti-HIV-1.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
本文编号：2198525
[Abstract]:Background:
In 2004, the rhesus monkey cDNA library was screened for TRIM5a, a cytoplasmic factor that inhibits HIV-1 infection. The rhesus monkey and macaque TRIM5a inhibited HIV-1 infection but not SIVmac. On the contrary, human TRIM5a had little inhibition on the above viruses and had the ability to inhibit Y retrovirus N-MLV. However, when individual amino acids are changed, human TRIM5a will have the same level of anti-HIV-1 function as rhesus monkey TRIM5a. Because of the existence of human anti-xenogeneic immune response, xenogeneic proteins in the human body are often quickly cleared, and their half-lives are also shorter, so our laboratory selected human TRIM5a (modified) for research.
Most of the previous studies on TRIM5a have been carried out at the gene level. For example, TRIM5a gene is transported into cells by liposome, viral vectors and so on to express in large quantities, thus changing the cell phenotype. So we have constructed the expression system of PTD-TRIM5a and TRIM5alpha H (R328-332) in E. coli, but the expression level is not high.
The penetrating efficiency of PTD-TRIM5alpha and PTD-TRIM5alpha H (R328-332) and the cytotoxicity and anti-HIV-1 ability of target cells were studied in this laboratory. The results showed that PTD-TRIM5alpha and PTD-TRIM5alpha H (R328-332) could enter the cells to play biological roles and have no toxicity to target cells under the action of transmembrane peptides. - 332) Human, but the laboratory has carried out several amino acid substitutions, so its safety needs to be further investigated, especially the toxic effects on normal human cells.
Up to now, the mechanism of TRIM5a restricting HIV-1 has become more and more clear. However, the mechanism of TRIM5a restricting HIV-1 replication is still unclear. Many problems remain unsolved, mostly at the speculative level. RELOCATION, MODIFICATION OR DEGRADATION? [1] Does TRIM5a contain a ubiquitin ligase subunit, or a similar SUMO (small ubiquitin-related modifier) transferase subunit? Is the reverse transcription process itself blocked by TRIM5a, or is it the result of degradation after DNA synthesis? What other proteins may TRIM5a bind to?
Research purposes:
1. The recombinant plasmid pET28a was transformed into E. coli strain BL21 (DE3) alpha, and the recombinant genes PTD-TRIM5alpha and PTD-TRIM5alphaH (R328-332) were confirmed to be expressed at gene level and protein level.
2. By comparing the expression of recombinant human TRIM5a chimeric plasmid constructed in our laboratory under different expression conditions, the optimal expression conditions of the target protein in E.coli were explored.
3. Natural human TRIM5a has low anti-HIV-1 ability. When the mutant TRIM5alpha H (R328-332) [i.e. I M (328), G Q (330), R P (332)] gene is transfected into cells by retroviral vectors, it has a better anti-HIV-1 effect, so the safety of the mutant TRIM5alpha H (R328-332) gene should be further investigated. Toxicity, this study further studies its toxicity to human normal cells.
4. In order to understand the mechanism of TRIM5a's anti-HIV-1 action, the subcellular sites of TRIM5a were observed by confocal microscopy.
5. to determine the relationship between ubiquitination of TRIM5 and its ability to resist HIV-1.
Research methods:
1. The recombinant gene PTD-TRIM5a and PTD-TRIM5alpha H (R328-332) were identified by enzyme digestion, PCR, gene sequencing, SDS-PAGE, Western-blotting and peptide fingerprint analysis.
2. SDS-PAGE electrophoresis was used to analyze the expression of recombinant protein in different temperature, IPTG concentration, induction time, induction period and culture medium, and to find out the optimum conditions for the maximum expression of the target protein in each factor.
3. The cytotoxicity of PTD-TRIM5a and PTD-TRIM5alpha H (R328-332) and its effect on cell proliferation were detected by Trypan blue exclusion test and SunBioTMAm-Blue assay.
4. The recombinant protein and nucleus were labeled with FITC and Hoechst fluorescence respectively, and the distribution of fluorescence was observed under confocal microscope to determine the subcellular structure of the recombinant protein.
5. the relationship between the ubiquitination of TRIM5 and its ability to resist HIV-1 was detected by ELISA.
Experimental results:
1. Target bands or peaks appeared at both gene level and protein level, and the recombinant genes PTD-TRIM5alpha and PTD-TRIM5alphaH (R328-332) were confirmed to be expressed.
2. The results showed that the expression of TRIM5a chimeric protein reached the maximum at 30 C, the induction concentration of IPTG was 0.5mmol/L, the induction time was 8 h, the OD value of bacterial fluid was 0.6, and the expression level of TRIM5a chimeric protein in TB medium was the highest.
3. The survival rate of 3T3 cells was above 83% when the recombinant protein was added to the cell samples, so the recombinant protein was safe and nontoxic.
4. analyzing the color of fluorescent dyes, it is known that the recombinant protein is only restricted to the cytoplasm and does not enter the nucleus.
5.When the concentrations of recombinant protein PTD-TRIM 5 alpha and PTD-TRIM 5 alpha H (R328-332) and PTD-TRIM 5 alphaH (R328-332) were 100 \\\ ml-1,10 \\\\, 10 \\\\\\\\, 1, 1\\\\\\\\\\\\\\\\\\\\\\\\\\\\1,164.3 pg.mL-1,88.6 pg.mL-1,51.4 pg.mL-1.
Conclusion:
1. The prokaryotic expression system of recombinant protein PTD-TRIM5alpha and PTD-TRIM5alpha H (R328-332) was successfully constructed with the efforts of our lab staff, and this study was identified at the level of gene and protein.
2. The prokaryotic expression system of recombinant protein PTD-TRIM5a and PTD-TRIM5alphaH (R328-332) was optimized, and the yield of recombinant protein was significantly increased.
3. The recombinant protein PTD-TRIM5a and PTD-TRIM5alphaH (R328-332) have been proved to be non-toxic to target cells in our laboratory. This study further proves that the recombinant protein is safe and non-toxic to human normal cells.
4. The anti-HIV-1 effect of recombinant protein PTD-TRIM5alpha and PTD-TRIM5alpha H (R328-332) was observed under confocal microscope by fluorescence labeling. The recombinant protein did not enter the nucleus and inhibited the replication of HIV-1 directly. Therefore, it was determined that the recombinant protein inhibited the life of HIV-1 before or after replication by blocking the replication of HIV-1. Activity process, so as to achieve the anti HIV-1 effect.
5. ELISA showed that the ubiquitination of recombinant protein PTD-TRIM5a and PTD-TRIM5alpha H (R328-332) was linearly related to the ability of anti-HIV-1.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
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