syncytin基因真核表达质粒pCMV-tag 2B-syncytin的构建

发布时间：2018-02-06 07:10

本文关键词： 人类内源性逆转录病毒质粒构建运动神经元病 HERVs syncytin　出处：《天津医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景与目的人类内源性逆转录病毒(Human endogenous retroviruses, HERVs)是几百万年前整合到人类基因组中,并以孟德尔方式遗传至今的逆转录病毒的残余物,约占整个基因组的8%。大部分HERVs在进化过程中由于突变、缺失等的积累,已丧失编码能力,但仍有少数HERVs的开放阅读框(Open reading frames, ORFs)被完整保留了下来。这些完整的ORFs可以编码逆转录病毒的蛋白,在一些特定的组织或分化发育的特定阶段表达,可能具有重要的生理意义,而在某些疾病情况下的异常表达提示其可能与疾病的发生发展相关。HERVs基因表达已被证实与多种神经精神疾病相关,如多发性硬化(multiple sclerosis, MS)和精神分裂症(schizophrenia),研究证实病人组织液和血清中含有多发性硬化相关逆转录病毒(Multiple sclerosis associated retrovirus, MSRV)和HERV-K基因表达。Oluwole等应用实时PCR技术检测了运动神经元病(motor neuron disease, MND)萎缩肌肉、未萎缩肌肉及正常对照肌肉组织中ERVWE1 env基因的表达,结果显示萎缩肌肉组织中ERVWE1 env mRNA水平比正常对照肌肉组织显著增高,比未萎缩肌肉组织也有增高。他们还检测了MND肌肉组织中SOD1基因的mRNA水平,结果显示在MND萎缩肌肉组织中SOD1 mRNA水平比未受累肌肉组织及健康对照明显增高,提示MND病变肌肉组织内存在过氧化损伤,支持syncytin的表达与氧化应激损伤具有相关性的观点。Oluwole等在国际上首次报道了MND组织中ERVWE1 env基因的异常转录,但其与MND可能的相关性及意义仍待进一步研究。本研究拟构建syncytin真核表达质粒,并通过脂质体法转染宫颈癌细胞株(Hela),并且采用RT-PCR检测syncytin mRNA水平。为进一步探讨ERVWE1 env基因的异常表达对运动神经元的损伤作用及机制奠定基础。方法①syncytin基因编码区的克隆：选取人胎盘组织提取总RNA,根据人syncytin基因编码区序列(核苷酸库中的编号：AF072506.2)设计引物,应用RT-PCR方法扩增人syncytin基因编码区全长,PCR产物经1%琼脂糖凝胶电泳鉴定大小。 ②syncytin基因真核表达质粒的构建：PCR产物与pCMV-tag 2B空载质粒经限制性内切酶BamHⅠ和EcoRⅠ酶切纯化后,确定连接反应体系,在T4连接酶作用下,室温5分钟快速连接。将连接产物转化Trans1-T1 phage Resisitant化学感受态细胞细胞,并接种于Ka+抗性琼脂培养皿,挑取阳性重组子。 ③重组质粒的鉴定：取阳性克隆菌液,进行直接菌液PCR法鉴定,然后对初步鉴定为阳性克隆的菌液提取质粒,应用限制性内切酶BamHⅠ和EcoRⅠ进行质粒的双酶切鉴定,并通过测序进一步鉴定。 ④转染人宫颈癌细胞株(Hela):转染前一天,以3×105个细胞/孔接种于6孔细胞培养板中,培养于无抗生素的生长培养基中,待细胞生长至80%细胞融合时转染,转染方法按Invitrogen公司提供的Lipofectamine 2000说明书进行。转染分两组,分别转染重组质粒pCMV-tag 2B-syncytin与空载体pCMV-tag 2B,转染后的细胞于37℃,5%CO2条件下培养。 ⑤检测人宫颈癌细胞(Hela) syncytin mRNA水平：提取Hela细胞总RNA,在M-MLV逆转录酶作用下,逆转录合成cDNA,再以逆转录合成的cDNA为模板进行实时荧光定量PCR检测,以β-actin作为为内参。结果①成功从人胎盘组织克隆了人syncytin基因编码区全长。 ②成功构建syncytin基因真核表达载体pCMV-tag 2B-syncytin,分别采用PCR及酶切鉴定,可得到相应大小的目的条带。 ③插入pCMV-tag 2B载体的syncytin基因序列分析与Genebank syncytin基因序列符合率为99%,不匹配碱基经遗传密码子表比对,均编码同一氨基酸。 ④重组质粒pCMV-tag 2B-syncytin转染至人宫颈癌细胞(Hela),实时荧光定量PCR检测syncytin mRNA水平,转染重组质粒pCMV-tag 2B-syncytin基因组syncytin mRNA水平比转染空载质粒pCMV-tag 2B组显著增高(P0.01),表明重组质粒pCMV-tag 2B-syncytin能成功转染入人宫颈癌细胞中并有效表达。结论成功构建了syncytin基因的真核表达质粒pCMV-tag 2B-syncytin,为进一步探讨syncytin的异常表达对运动神经元的可能损伤作用及机制奠定坚实基础。
[Abstract]:Background and objective: human endogenous retrovirus (Human endogenous, retroviruses, HERVs) is integrated into the millions of years ago in the human genome, residue and Mendel has genetic retrovirus, accounting for the entire genome 8%. most of the HERVs in the process of evolution due to mutations, such as lack of accumulation, has lost the encoding ability, but still there are a few open reading frame of HERVs (Open reading frames, ORFs) is completely preserved. These can complete ORFs encoding retroviral protein expression in specific tissues or differentiation of the particular stage may have important physiological significance, and in the case of certain diseases of abnormal expression suggests that it may be with the occurrence and development of disease related.HERVs gene expression has been demonstrated to be associated with a variety of neuropsychiatric disorders, such as multiple sclerosis (multiple sclerosis, MS) and spirit Schizophrenia (schizophrenia), the research confirmed the presence of multiple sclerosis associated retroviral patient tissue fluid and serum (Multiple sclerosis associated retrovirus, MSRV) and HERV-K gene expression in real-time application of PCR.Oluwole detection of motor neuron disease (motor neuron, disease, MND) muscle atrophy, muscle atrophy without expression and normal muscle tissue in the ERVWE1 env gene, the results showed that ERVWE1 env mRNA level in muscle atrophy was significantly higher than the normal control of muscle tissue, compared to unaffected muscles also increased. They also tested SOD1 MND mRNA in muscle tissue mRNA levels, the results are displayed in the MND SOD1 mRNA level in muscle atrophy was significantly higher than unaffected muscles tissue and healthy controls, suggesting that MND lesions in memory of muscle tissue peroxidation injury, expression of support for syncytin and oxidative stress have Close view of.Oluwole in international reporting for the first time the abnormal transcription of MND env gene in ERVWE1 and MND, but its possible correlation and significance needs further study.
The aim of this study is to construct syncytin eukaryotic expression plasmid and transfect cervical cancer cell line (Hela) by liposome method, and detect the level of syncytin mRNA by RT-PCR. It will lay a foundation for further discussing the abnormal expression of ERVWE1 env on motor neuron damage and its mechanism.
Cloning method of syncytin gene encoding region: human placenta extract total RNA, according to the sequence of syncytin gene encoding region (nucleotide base number: AF072506.2) primers were designed using the method of RT-PCR amplification of full-length human syncytin gene encoding region, PCR products were tested by 1% agarose gel electrophoresis identification.
The construction of eukaryotic expression plasmid of syncytin: PCR products and pCMV-tag empty plasmid 2B by restriction endonuclease BamH I and EcoR I restriction enzyme after purification, determine the connection in the reaction system, T4 ligase, at room temperature for 5 minutes. The quick connect products were transformed Trans1-T1 phage Resisitant chemically competent cells, and inoculated Ka+ resistant agar plate, then the positive recombinants.
(3) identification of recombinant plasmid: positive clone bacteria liquid was identified by direct bacterial liquid PCR method, then plasmid was identified as positive clone, then restriction enzyme BamH I and EcoR I were used to identify plasmid double enzyme, and further identified by sequencing.
The transfection of human cervical carcinoma cell lines (Hela): the day before transfection, with 3 x 105 cells were seeded in 6 well cell culture plates and cultured in antibiotic free growth medium, when the cells grow to 80% cell fusion transfection, transfection method provided by Invitrogen company of the Lipofectamine 2000 specification the transfection was divided into two groups, were transfected with the recombinant plasmid pCMV-tag 2B-syncytin and empty vector pCMV-tag 2B and transfected cells at 37 deg.c, under the condition of 5%CO2 culture.
The detection of human cervical carcinoma cell line (Hela) syncytin mRNA level: Hela extraction of total cellular RNA in M-MLV reverse transcriptase. CDNA was synthesized by reverse transcriptase, then the synthesis of cDNA was amplified by real-time fluorescence quantitative PCR detection, with beta -actin as for reference.
Results 1. The full length of the human syncytin gene coding region was successfully cloned from the human placental tissue.
(2) the syncytin gene eukaryotic expression vector pCMV-tag 2B-syncytin was successfully constructed, and the target bands of corresponding size could be obtained by using PCR and enzyme digestion respectively.
(3) the sequence analysis of syncytin gene inserted into pCMV-tag 2B vector and Genebank syncytin gene sequence coincidence rate is 99%. The mismatched bases are encoded by the same amino acid by genetic codon comparison.
The recombinant plasmid pCMV-tag 2B-syncytin was transfected into human cervical carcinoma cells (Hela), syncytin mRNA real time fluorescence quantitative PCR detection level of recombinant plasmid pCMV-tag 2B-syncytin genomic syncytin mRNA levels were significantly higher than those transfected plasmid pCMV-tag 2B group (P0.01), the recombinant plasmid pCMV-tag 2B-syncytin was successfully transfected into human cervical carcinoma cells and effective expression..
Conclusion the eukaryotic expression plasmid pCMV-tag 2B-syncytin of syncytin gene has been successfully constructed, which lays a solid foundation for further exploring the potential role of abnormal expression of syncytin on motor neurons and its mechanism.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R744;R346

【共引文献】