重组人端粒酶逆转录酶基因表达载体的构建及检测端粒长度新方法初探
发布时间：2018-09-11 13:07
【摘要】：第一章人端粒酶催化亚基(hTERT)的研究现状和研究进展(文献综述) 人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)基因是端粒酶的催化亚基,负责在染色体末端添加端粒重复序列,在维持细胞永生化中起重要作用。为探索过表达外源性hTERT基因转染对细胞生物学作用,本研究以人脐静脉血管内皮细胞(hUVEC)为研究对象,通过真核转染及病毒载体介导的基因转移技术促进hTERT基因在hUVEC细胞中过表达,观察其对hUVEC细胞增殖作用和端粒酶活性的影响。现综合近年来的文献,对人端粒酶催化亚基的研究现状作一简要综述。 第二章过表达人端粒酶逆转录酶促进人脐静脉血管内皮细胞增殖 目的:构建含人端粒酶逆转录酶(hTERT)基因的真核表达载体并转染人脐静脉血管内皮细胞(hUVEC),探索转染后hTERT基因的表达及对细胞功能和生长的影响。 方法:携带人端粒酶逆转录酶(hTERT)基因的重组质粒(pEGFP-C1-hTERT)是利用已有的PCI-neo-hTERT和pEGFP-C1质粒,通过酶切连接后,DNA测序验证了重组质粒pEGFP-C1-hTERT的准确性。将pEGFP-C1-hTERT真核表达载体通过脂质体2000转染到hUVEC中,通过RT-PCR、免疫组化、PCR-ELISA法和MTT检测细胞端粒酶基因表达和活性变化与细胞生长增殖情况。 结果:所构建pEGFP-C1-hTERT真核表达载体结构正确并能够在真核细胞中表达。转染后细胞可见报告基因GFP的表达;MTT实验可见转染hTERT基因组72 h后细胞增殖速度快于未转染组及空载体组;RT-PCR、免疫组化及TRAP-PCR-ELISA法检测转染后的细胞,结果发现hTERT mRNA的表达、端粒酶活性均明显增强。 结论:本研究成功构建了pEGFP-C1-hTERT真核表达载体并能够在真核细胞中表达,过表达的hTERT基因提高了血管内皮细胞端粒酶的活性和增殖能力,初步证实端粒酶活性与细胞增殖活性密切相关,为进一步构建永生化细胞系奠定基础。 第三章构建携带重组人端粒酶逆转录酶第三代慢病毒载体及其病毒包装鉴定 目的:构建携带人端粒酶逆转录酶(hTERT)基因的慢病毒表达载体及探索高滴度第三代慢病毒包装体系的建立,并观察hTERT基因调控表达。 方法:用内切酶将hTERT基因从已有质粒PCI-neo-hTERT上切下,插入慢病毒载体pCDH-copGFP中构建慢病毒表达质粒pCDH-hTERT,通过双酶切鉴定、DNA测序分析验证人端粒酶逆转录酶基因(hTERT)片段的准确性后,将pCDH- hTERT、pCDH-PACK-GAG、pCDH-PACK-REV和VSV-G共转染包装细胞293T,浓缩上清并测定病毒滴度获得重组慢病毒,并进行PCR及293T中hTERT蛋白的表达鉴定重组慢病毒的包装。重组慢病毒再感染靶细胞hUVEC,通过检测标记蛋白-绿色荧光蛋白、hTERT蛋白表达和端粒酶活性进一步验证pCDH-hTERT在细胞中表达。 结果:pCDH-hTERT携带正确hTERT基因,将其与包装质粒共转染293T细胞能产生重组病毒;病毒基因组PCR证实hTERT基因插入,感染后293T可检测到hTERT蛋白的表达;目的基因hTERT能被重组慢病毒高效地转导入靶细胞并稳定表达,荧光显微镜下可直接观察GFP;RT-PCR法、Western blotting法及TRAP-PCR-ELISA法能检测感染后的细胞,结果发现hTERT mRNA的表达、hTERT蛋白的表达及端粒酶活性明显增强。 结论:本研究成功构建了第三代慢病毒表达载体pCDH-hTERT,并获得高效的重组慢病毒,将外源hTERT基因转导入靶细胞重建端粒酶活性,为构建永生化细胞系奠定基础。 第四章广西长寿物种端粒长度的检测与纳米金标记的巯基寡聚核苷酸探针快速灵敏检测端粒长度新方法的初探 目的:应用Southern杂交技术对不同年龄段不同长寿物种端粒长度进行检测,从而了解Southern杂交对端粒长度检测的利弊,评估其是否适用于其他长寿物种的检测,探讨建立检测针对不同物种端粒长度新方法的必要性,并初步探索利用纳米金技术建立快速灵敏检测端粒长度新方法可行性。 方法:选择广西常见的长寿物种草龟、眼镜蛇作为研究对象,应用Southern杂交技术进行检测不同年龄段不同长寿物种端粒长度,对检测端粒长度进行比较研究。以已知三种不同长度的端粒重复片段为标准品,初步探索将纳米金的共振散射效应和纳米金标记核酸探针结合起来建立一种测定端粒长度的新方法。通过制备粒径约10纳米的金纳米颗粒,用于标记针对人端粒重复序列的共振散射光谱探针5’-(CCCTAA)5(CH2)3SH-3’,对标记纳米金生物探针上寡核苷酸连接及与样品端粒杂交反应体系条件进行优化,探索利用纳米金技术建立快速灵敏检测端粒长度新方法的可行性。 结果:所应用Southern杂交技术进行检测不同年龄段不同长寿物种端粒长度特异性及灵敏性均不高,各组间比较均无统计学意义。并成功完成标记纳米金生物探针上寡核苷酸的连接,通过优化了杂交反应体系中缓冲溶液的pH、AussDNA浓度、NaCl浓度、超声波辐照时间等四个主要反应条件的影响,杂交后信号波动较大,但仍未找到适合的杂交的反应条件,尚未能建立起稳定的纳米金探针检测端粒长度的新方法。 结论:Southern杂交技术采用针对人端粒重复片段特定探针,不适合应用于其他长寿物种的检测,故需建立快速灵敏检测针对不同物种端粒长度新方法。新方法中标记好纳米金生物探针与样品端粒杂交反应体系是实验的一大难题,如何解决和建立纳米金生物探针与样品端粒杂交反应体系将是后续的实验的首要解决问题。
[Abstract]:Chapter one: research status and research progress of human telomerase catalytic subunit (hTERT) (literature review)
Human telomerase reverse transcriptase (hTERT) gene is a catalytic subunit of telomerase, which is responsible for adding telomere repeats at the end of chromosome and plays an important role in maintaining cell immortality. To explore the effect of exogenous hTERT gene transfection on cell biology, this study was conducted in human umbilical vein. Dermal cells (hUVEC) were studied to promote the overexpression of hTERT gene in hUVEC cells by eukaryotic transfection and virus vector-mediated gene transfer. The effects of hTERT gene on the proliferation and telomerase activity of hUVEC cells were observed.
Second chapter overexpression of human telomerase reverse transcriptase promotes proliferation of human umbilical vein endothelial cells
AIM: To construct an eukaryotic expression vector containing human telomerase reverse transcriptase (hTERT) gene and transfect it into human umbilical vein endothelial cells (hUVEC) to investigate the expression of hTERT gene and its effect on cell function and growth.
METHODS: Recombinant plasmid carrying human telomerase reverse transcriptase (hTERT) gene (pEGFP-C1-hTERT) was constructed by using the existing plasmids PCI-neo-hTERT and pEGFP-C1. DNA sequencing confirmed the accuracy of the recombinant plasmid pEGFP-C1-hTERT. Epidemic histochemistry, PCR-ELISA and MTT assay were used to detect the expression and activity of telomerase gene and cell proliferation.
Results: The constructed eukaryotic expression vector of pEGFP-C1-hTERT was constructed correctly and could express in eukaryotic cells.GFP expression was observed in the transfected cells.MTT assay showed that the proliferation rate of the transfected cells was faster than that of the untransfected and empty vectors 72 hours after transfection.RT-PCR, immunohistochemistry and TRAP-PCR-ELISA were used to detect the transfected cells. It was found that the expression of hTERT mRNA and telomerase activity were significantly enhanced.
Conclusion: pEGFP-C1-hTERT eukaryotic expression vector was successfully constructed and expressed in eukaryotic cells. Overexpression of hTERT gene enhanced telomerase activity and proliferation ability of vascular endothelial cells. It was preliminarily confirmed that telomerase activity was closely related to cell proliferation activity and laid a foundation for further construction of immortalized cell lines.
The third chapter is to construct a third generation lentiviral vector carrying recombinant human telomerase reverse transcriptase and its viral packaging identification.
Objective: To construct lentiviral expression vector carrying human telomerase reverse transcriptase (hTERT) gene and explore the establishment of high titer third generation lentiviral packaging system, and to observe the regulation of hTERT gene expression.
METHODS: The hTERT gene was cut from the existing plasmid PCI-neo-hTERT by endonuclease and inserted into the lentiviral vector pCDH-copGFP to construct the lentiviral expression plasmid pCDH-hTERT. The accuracy of the fragment of human telomerase reverse transcriptase gene (hTERT) was verified by double enzyme digestion and DNA sequencing analysis. Then pCDH-hTERT, pCDH-PACK-GAG, pCDH-PACK-REV and VSV-G were co-constructed. The recombinant lentivirus was obtained by transfection of 293T into packaging cells, concentration of supernatant and determination of viral titer. The recombinant lentivirus was identified by PCR and expression of hTERT protein in 293T. Expression in cells.
Results: pCDH-hTERT carried the correct hTERT gene and transfected it with the packaging plasmid into 293T cells to produce recombinant virus. Viral genome PCR confirmed that hTERT gene was inserted into 293T cells and the expression of hTERT protein was detected after infection. The target gene hTERT could be transfected into target cells efficiently and stably expressed by recombinant lentivirus, and could be directly expressed under fluorescence microscope. GFP, RT-PCR, Western blotting and TRAP-PCR-ELISA were used to detect the expression of hTERT mRNA, hTERT protein and telomerase activity.
CONCLUSION: The third generation lentiviral expression vector pCDH-hTERT was successfully constructed and highly efficient recombinant lentiviruses were obtained. The foreign hTERT gene was transfected into target cells to reconstruct telomerase activity and lay a foundation for the construction of immortalized cell lines.
CHAPTER IV DETECTION OF TERMINAL LENGTH IN LONG-LIFE SPECIES OF GUANGXI PROVINCE
Objective: To investigate the advantages and disadvantages of Southern hybridization for telomere length detection in different age groups of longevity species, to assess whether it is suitable for other longevity species, to explore the necessity of establishing a new method for telomere length detection in different species, and to explore the use of Na. It is feasible to establish a fast and sensitive method for telomere length detection.
METHODS: The telomere lengths of common longevity turtles and cobras in Guangxi were measured by Southern hybridization technique, and the telomere lengths of different longevity species in different ages were compared. The resonance scattering of gold nanoparticles was preliminarily explored using three telomere repeats with different lengths as standard samples. A new method for determining telomere length was developed by combining the effect with gold nanoparticle labeled nucleic acid probes. The gold nanoparticles with a diameter of about 10 nm were prepared to label the resonance scattering probe 5'-(CCCTAA) 5 (CH2) 3SH-3', to connect the oligonucleotides on the labeled gold nanoprobe and to the end of the sample. The conditions of particle hybridization reaction system were optimized to explore the feasibility of establishing a new method for rapid and sensitive detection of telomere length by nanogold technology.
Results: The specificity and sensitivity of telomere length in different age groups were not high, and there was no significant difference among groups. The ligation of oligonucleotides on the labeled gold nanoprobe was successfully completed. The pH of buffer solution, AussDNA concentration, NaC in the hybridization reaction system were optimized. L concentration, ultrasonic irradiation time and other four main reaction conditions, the signal fluctuated greatly after hybridization, but no suitable hybridization reaction conditions have been found, and no stable nano-gold probe has been established to detect telomere length.
CONCLUSION: Southern hybridization technique using specific probes for human telomere repeat fragments is not suitable for other long-lived species, so it is necessary to establish a new method for rapid and sensitive detection of telomere length for different species. It will be the first problem to establish the reaction system of gold nanoprobe and telomere hybridization.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
，
本文编号：2236768
[Abstract]:Chapter one: research status and research progress of human telomerase catalytic subunit (hTERT) (literature review)
Human telomerase reverse transcriptase (hTERT) gene is a catalytic subunit of telomerase, which is responsible for adding telomere repeats at the end of chromosome and plays an important role in maintaining cell immortality. To explore the effect of exogenous hTERT gene transfection on cell biology, this study was conducted in human umbilical vein. Dermal cells (hUVEC) were studied to promote the overexpression of hTERT gene in hUVEC cells by eukaryotic transfection and virus vector-mediated gene transfer. The effects of hTERT gene on the proliferation and telomerase activity of hUVEC cells were observed.
Second chapter overexpression of human telomerase reverse transcriptase promotes proliferation of human umbilical vein endothelial cells
AIM: To construct an eukaryotic expression vector containing human telomerase reverse transcriptase (hTERT) gene and transfect it into human umbilical vein endothelial cells (hUVEC) to investigate the expression of hTERT gene and its effect on cell function and growth.
METHODS: Recombinant plasmid carrying human telomerase reverse transcriptase (hTERT) gene (pEGFP-C1-hTERT) was constructed by using the existing plasmids PCI-neo-hTERT and pEGFP-C1. DNA sequencing confirmed the accuracy of the recombinant plasmid pEGFP-C1-hTERT. Epidemic histochemistry, PCR-ELISA and MTT assay were used to detect the expression and activity of telomerase gene and cell proliferation.
Results: The constructed eukaryotic expression vector of pEGFP-C1-hTERT was constructed correctly and could express in eukaryotic cells.GFP expression was observed in the transfected cells.MTT assay showed that the proliferation rate of the transfected cells was faster than that of the untransfected and empty vectors 72 hours after transfection.RT-PCR, immunohistochemistry and TRAP-PCR-ELISA were used to detect the transfected cells. It was found that the expression of hTERT mRNA and telomerase activity were significantly enhanced.
Conclusion: pEGFP-C1-hTERT eukaryotic expression vector was successfully constructed and expressed in eukaryotic cells. Overexpression of hTERT gene enhanced telomerase activity and proliferation ability of vascular endothelial cells. It was preliminarily confirmed that telomerase activity was closely related to cell proliferation activity and laid a foundation for further construction of immortalized cell lines.
The third chapter is to construct a third generation lentiviral vector carrying recombinant human telomerase reverse transcriptase and its viral packaging identification.
Objective: To construct lentiviral expression vector carrying human telomerase reverse transcriptase (hTERT) gene and explore the establishment of high titer third generation lentiviral packaging system, and to observe the regulation of hTERT gene expression.
METHODS: The hTERT gene was cut from the existing plasmid PCI-neo-hTERT by endonuclease and inserted into the lentiviral vector pCDH-copGFP to construct the lentiviral expression plasmid pCDH-hTERT. The accuracy of the fragment of human telomerase reverse transcriptase gene (hTERT) was verified by double enzyme digestion and DNA sequencing analysis. Then pCDH-hTERT, pCDH-PACK-GAG, pCDH-PACK-REV and VSV-G were co-constructed. The recombinant lentivirus was obtained by transfection of 293T into packaging cells, concentration of supernatant and determination of viral titer. The recombinant lentivirus was identified by PCR and expression of hTERT protein in 293T. Expression in cells.
Results: pCDH-hTERT carried the correct hTERT gene and transfected it with the packaging plasmid into 293T cells to produce recombinant virus. Viral genome PCR confirmed that hTERT gene was inserted into 293T cells and the expression of hTERT protein was detected after infection. The target gene hTERT could be transfected into target cells efficiently and stably expressed by recombinant lentivirus, and could be directly expressed under fluorescence microscope. GFP, RT-PCR, Western blotting and TRAP-PCR-ELISA were used to detect the expression of hTERT mRNA, hTERT protein and telomerase activity.
CONCLUSION: The third generation lentiviral expression vector pCDH-hTERT was successfully constructed and highly efficient recombinant lentiviruses were obtained. The foreign hTERT gene was transfected into target cells to reconstruct telomerase activity and lay a foundation for the construction of immortalized cell lines.
CHAPTER IV DETECTION OF TERMINAL LENGTH IN LONG-LIFE SPECIES OF GUANGXI PROVINCE
Objective: To investigate the advantages and disadvantages of Southern hybridization for telomere length detection in different age groups of longevity species, to assess whether it is suitable for other longevity species, to explore the necessity of establishing a new method for telomere length detection in different species, and to explore the use of Na. It is feasible to establish a fast and sensitive method for telomere length detection.
METHODS: The telomere lengths of common longevity turtles and cobras in Guangxi were measured by Southern hybridization technique, and the telomere lengths of different longevity species in different ages were compared. The resonance scattering of gold nanoparticles was preliminarily explored using three telomere repeats with different lengths as standard samples. A new method for determining telomere length was developed by combining the effect with gold nanoparticle labeled nucleic acid probes. The gold nanoparticles with a diameter of about 10 nm were prepared to label the resonance scattering probe 5'-(CCCTAA) 5 (CH2) 3SH-3', to connect the oligonucleotides on the labeled gold nanoprobe and to the end of the sample. The conditions of particle hybridization reaction system were optimized to explore the feasibility of establishing a new method for rapid and sensitive detection of telomere length by nanogold technology.
Results: The specificity and sensitivity of telomere length in different age groups were not high, and there was no significant difference among groups. The ligation of oligonucleotides on the labeled gold nanoprobe was successfully completed. The pH of buffer solution, AussDNA concentration, NaC in the hybridization reaction system were optimized. L concentration, ultrasonic irradiation time and other four main reaction conditions, the signal fluctuated greatly after hybridization, but no suitable hybridization reaction conditions have been found, and no stable nano-gold probe has been established to detect telomere length.
CONCLUSION: Southern hybridization technique using specific probes for human telomere repeat fragments is not suitable for other long-lived species, so it is necessary to establish a new method for rapid and sensitive detection of telomere length for different species. It will be the first problem to establish the reaction system of gold nanoprobe and telomere hybridization.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346
，
本文编号：2236768
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