PTD-Trim5α和PTD-Trim5αH（R328-332）在大腸桿菌中的表達優(yōu)化及其作用機制研究
發(fā)布時間：2018-08-23 08:46
【摘要】： 前期工作背景： 在2004年,對恒河猴的cDNA文庫進行篩選得到TRIM5α,一種能夠抑制HIV-1感染的細胞質(zhì)因子。恒河猴和短尾猴的TRIM5α能夠抑制HIV-1感染而不能抑制SIVmac,與此相反,人的TRIM5α對上述病毒幾乎沒有什么抑制作用,具有能力抑制Y逆轉(zhuǎn)錄病毒N-MLV。然而,當(dāng)有個別氨基酸改變時,人源的TRIM5α將具有和恒河猴TRIM5α相似水平的抗HIV-1功能。由于人抗異種動物的免疫應(yīng)答反應(yīng)的存在,異種蛋白在人體內(nèi)往往被快速清除,其半衰期也較短,因此本實驗室選擇人源的TRIM5α(改構(gòu)體)進行研究。 以往關(guān)于TRIM5α的絕大多數(shù)研究,主要圍繞在基因水平上進行的,例如,通過脂質(zhì)體、病毒載體等結(jié)構(gòu)將TRIM5α基因轉(zhuǎn)運進入細胞使之大量表達,從而改變細胞的表型。在實際操作過程中,往往存在可操作性差,轉(zhuǎn)運效率低,穩(wěn)定性不好,生物毒性作用的缺點。所以本實驗室前期已經(jīng)構(gòu)建出PTD-TRIM5α和TRIM5αH (R328-332)的大腸桿菌表達系統(tǒng),但是其表達量不高。 本實驗室對PTD-TRIM5α和PTD-TRIM5αH(R328-332)的穿膜效率、對靶細胞的細胞毒性和抗HIV-1的能力進行了研究,表明PTD-TRIM5α和PTD-TRIM5αH(R328-332)在穿膜肽的作用下,能夠進入細胞發(fā)揮生物學(xué)作用,對靶細胞也無毒性。雖然PTD-TRIM5α和PTD-TRIM5αH(R328-332)是人源的,但是本實驗室進行了幾個氨基酸的置換,所以其安全性有必要進一步考察,尤其是對正常人體細胞的毒性作用。 到目前為止,TRIM5α限制HIV-1的機制已越來越清楚,但是,人們對于TRIM5α限制HIV-1復(fù)制的作用機制尚不完全清楚,許多問題還沒有解決,大多停留在推測的水平。本實驗室已經(jīng)得出它直接與HIV-1的核衣殼蛋白gag結(jié)合,但它是否會導(dǎo)致核衣殼的重新定位、修飾或降解?[1]TRIM5α是否包含有一個泛素連接酶的亞單位,或者一個相似作用的SUMO (small ubiquitin-related modifier)轉(zhuǎn)移酶的亞基?逆轉(zhuǎn)錄過程本身被TRIM5α阻斷,還是DNA合成后降解的結(jié)果?TRIM5α還可能與其他什么蛋白結(jié)合? 研究目的： 1.將課題組前期構(gòu)建的重組質(zhì)粒pET28a轉(zhuǎn)化大腸菌株BL21(DE3)α從基因水平上和蛋白質(zhì)水平上,確定重組基因PTD-TRIM5α和PTD-TRIM5αH(R328-332)得到表達。 2.通過對比本實驗室構(gòu)建的人TRIM5α嵌合體重組質(zhì)粒在各種不同表達條件下的表達量,從而探索出目的蛋白在大腸桿菌中的最佳表達條件。 3.天然人源的TRIM5α抗HIV-1的能力比較低,當(dāng)突變的TRIM5αH(R328-332)[即I→M(328)、G→Q(330)、R→P(332)]基因用逆轉(zhuǎn)錄病毒載體轉(zhuǎn)染細胞后,具有較好的抑制HIV-1的作用,本實驗室進行了上述突變,所以其安全性有必要進一步考察。對靶細胞的細胞毒性,本研究進一步研究其對人體正常細胞的毒性。 4.本實驗室已經(jīng)得出它直接與HIV-1的核衣殼蛋白gag結(jié)合,為深入了解TRIM5α的抗HIV-1的機制,運用共聚焦顯微鏡觀察TRIM5α作用的亞細胞部位。 5.確定TRIM5α的泛素化與其抗HIV-1的能力的關(guān)系。 研究方法： 1.在基因水平上應(yīng)用酶切鑒定、PCR鑒定、基因測序,在蛋白水平上利用SDS-PAGE、Western-blotting、肽圖指紋圖譜鑒定,進行重組基因PTD-TRIM5α和PTD-TRIM5αH (R328-332)表達的鑒定。 2.運用SDS-PAGE電泳分析重組蛋白在不同溫度、IPTG的濃度、誘導(dǎo)時長、誘導(dǎo)時期與培養(yǎng)基的表達量,分別找出各個因素的目的蛋白表達量最大的最適條件。 3.應(yīng)用臺盼藍排斥實驗和SunBioTMAm-Blue檢測比色法檢測PTD-TRIM5α和PTD-TRIM5αH(R328-332)的細胞毒性和對細胞增殖的影響。 4.重組蛋白和細胞核分別用FITC和Hoechst熒光標(biāo)記,在共聚焦顯微鏡觀察下觀察熒光的分布情況,從而確定重組蛋白作用的亞細胞結(jié)構(gòu)部位。 5.采用ELISA方法檢測TRIM5的泛素化與其抗HIV-1的能力的關(guān)系。 實驗結(jié)果： 1.在基因水平上和蛋白質(zhì)水平上,都出現(xiàn)了目的條帶或目標(biāo)峰,確定重組基因PTD-TRIM5α和PTD-TRIM5αH (R328-332)得到表達。 2.通過分析比較,重組質(zhì)粒在30℃、IPTG的誘導(dǎo)濃度為0.5mmol/L、誘導(dǎo)時長為8h、菌液的OD值為0.6以及在TB培養(yǎng)基上培養(yǎng)人TRIM5α嵌合體蛋白表達量達到最大。 3.在細胞樣品中加入重組蛋白,3T3細胞的存活率都在83%以上,所以重組蛋白是安全無毒性的。 4.分析熒光染料的顏色可知,重組蛋白只限定在細胞質(zhì)內(nèi),沒有進入細胞核內(nèi)。 5.當(dāng)重組蛋白PTD-TRIM5α和PTD-TRIM5αH(R328-332)的濃度分別為100μg·ml-1、10μg-mL-1、1μg-mL-1、0.1μg-1、0.01μg-mL-1時,UBPL濃度的濃度分別為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。 結(jié)論： 1.在本實驗室人員地努力下,成功地構(gòu)建了表達重組蛋白PTD-TRIM5α和PTD-TRIM5αH(R328-332)的大腸桿菌原核表達系統(tǒng),并且本研究在基因水平和蛋白質(zhì)水平上得到鑒定。 2.對重組蛋白PTD-TRIM5α和PTD-TRIM5αH(R328-332)的大腸桿菌原核表達系統(tǒng)的表達條件進行了優(yōu)化,使得重組蛋白質(zhì)得率有顯著地提高。 3.之前本實驗室已經(jīng)證明了重組蛋白PTD-TRIM5α和PTD-TRIM5αH(R328-332)對靶細胞無毒性,本研究進一步證明了重組蛋白對人體正常細胞是安全無毒性作用。 4.通過熒光標(biāo)記,在共聚焦顯微鏡下觀察重組蛋白PTD-TRIM5α和PTD-TRIM5αH (R328-332)的抗HIV-1的作用限定在細胞質(zhì)內(nèi),不進入細胞核,重組蛋白沒有進入細胞核直接抑制HIV-1的復(fù)制,故可確定重組蛋白是通過阻礙HIV-1復(fù)制前的或復(fù)制后的生命活動過程,從而達到抗HIV-1的作用。 5.通過ELISA法檢測發(fā)現(xiàn),重組蛋白PTD-TRIM5α和PTD-TRIM5αH (R328-332)的泛素化作用與抗HIV-1能力呈一定地線性關(guān)系。由此可知,重組蛋白有可能是通過促進對特異性病毒蛋白質(zhì)的水解,達到抗病毒作用。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378
本文編號：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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378
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