本文關(guān)鍵詞： 人類內(nèi)源性逆轉(zhuǎn)錄病毒 質(zhì)粒構(gòu)建 運動神經(jīng)元病 HERVs syncytin　出處：《天津醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 背景與目的人類內(nèi)源性逆轉(zhuǎn)錄病毒(Human endogenous retroviruses, HERVs)是幾百萬年前整合到人類基因組中,并以孟德爾方式遺傳至今的逆轉(zhuǎn)錄病毒的殘余物,約占整個基因組的8%。大部分HERVs在進化過程中由于突變、缺失等的積累,已喪失編碼能力,但仍有少數(shù)HERVs的開放閱讀框(Open reading frames, ORFs)被完整保留了下來。這些完整的ORFs可以編碼逆轉(zhuǎn)錄病毒的蛋白,在一些特定的組織或分化發(fā)育的特定階段表達,可能具有重要的生理意義,而在某些疾病情況下的異常表達提示其可能與疾病的發(fā)生發(fā)展相關(guān)。HERVs基因表達已被證實與多種神經(jīng)精神疾病相關(guān),如多發(fā)性硬化(multiple sclerosis, MS)和精神分裂癥(schizophrenia),研究證實病人組織液和血清中含有多發(fā)性硬化相關(guān)逆轉(zhuǎn)錄病毒(Multiple sclerosis associated retrovirus, MSRV)和HERV-K基因表達。Oluwole等應(yīng)用實時PCR技術(shù)檢測了運動神經(jīng)元病(motor neuron disease, MND)萎縮肌肉、未萎縮肌肉及正常對照肌肉組織中ERVWE1 env基因的表達,結(jié)果顯示萎縮肌肉組織中ERVWE1 env mRNA水平比正常對照肌肉組織顯著增高,比未萎縮肌肉組織也有增高。他們還檢測了MND肌肉組織中SOD1基因的mRNA水平,結(jié)果顯示在MND萎縮肌肉組織中SOD1 mRNA水平比未受累肌肉組織及健康對照明顯增高,提示MND病變肌肉組織內(nèi)存在過氧化損傷,支持syncytin的表達與氧化應(yīng)激損傷具有相關(guān)性的觀點。Oluwole等在國際上首次報道了MND組織中ERVWE1 env基因的異常轉(zhuǎn)錄,但其與MND可能的相關(guān)性及意義仍待進一步研究。 本研究擬構(gòu)建syncytin真核表達質(zhì)粒,并通過脂質(zhì)體法轉(zhuǎn)染宮頸癌細胞株(Hela),并且采用RT-PCR檢測syncytin mRNA水平。為進一步探討ERVWE1 env基因的異常表達對運動神經(jīng)元的損傷作用及機制奠定基礎(chǔ)。 方法①syncytin基因編碼區(qū)的克�。哼x取人胎盤組織提取總RNA,根據(jù)人syncytin基因編碼區(qū)序列(核苷酸庫中的編號：AF072506.2)設(shè)計引物,應(yīng)用RT-PCR方法擴增人syncytin基因編碼區(qū)全長,PCR產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳鑒定大小。 ②syncytin基因真核表達質(zhì)粒的構(gòu)建：PCR產(chǎn)物與pCMV-tag 2B空載質(zhì)粒經(jīng)限制性內(nèi)切酶BamHⅠ和EcoRⅠ酶切純化后,確定連接反應(yīng)體系,在T4連接酶作用下,室溫5分鐘快速連接。將連接產(chǎn)物轉(zhuǎn)化Trans1-T1 phage Resisitant化學(xué)感受態(tài)細胞細胞,并接種于Ka+抗性瓊脂培養(yǎng)皿,挑取陽性重組子。 ③重組質(zhì)粒的鑒定：取陽性克隆菌液,進行直接菌液PCR法鑒定,然后對初步鑒定為陽性克隆的菌液提取質(zhì)粒,應(yīng)用限制性內(nèi)切酶BamHⅠ和EcoRⅠ進行質(zhì)粒的雙酶切鑒定,并通過測序進一步鑒定。 ④轉(zhuǎn)染人宮頸癌細胞株(Hela):轉(zhuǎn)染前一天,以3×105個細胞/孔接種于6孔細胞培養(yǎng)板中,培養(yǎng)于無抗生素的生長培養(yǎng)基中,待細胞生長至80%細胞融合時轉(zhuǎn)染,轉(zhuǎn)染方法按Invitrogen公司提供的Lipofectamine 2000說明書進行。轉(zhuǎn)染分兩組,分別轉(zhuǎn)染重組質(zhì)粒pCMV-tag 2B-syncytin與空載體pCMV-tag 2B,轉(zhuǎn)染后的細胞于37℃,5%CO2條件下培養(yǎng)。 ⑤檢測人宮頸癌細胞(Hela) syncytin mRNA水平：提取Hela細胞總RNA,在M-MLV逆轉(zhuǎn)錄酶作用下,逆轉(zhuǎn)錄合成cDNA,再以逆轉(zhuǎn)錄合成的cDNA為模板進行實時熒光定量PCR檢測,以β-actin作為為內(nèi)參。 結(jié)果①成功從人胎盤組織克隆了人syncytin基因編碼區(qū)全長。 ②成功構(gòu)建syncytin基因真核表達載體pCMV-tag 2B-syncytin,分別采用PCR及酶切鑒定,可得到相應(yīng)大小的目的條帶。 ③插入pCMV-tag 2B載體的syncytin基因序列分析與Genebank syncytin基因序列符合率為99%,不匹配堿基經(jīng)遺傳密碼子表比對,均編碼同一氨基酸。 ④重組質(zhì)粒pCMV-tag 2B-syncytin轉(zhuǎn)染至人宮頸癌細胞(Hela),實時熒光定量PCR檢測syncytin mRNA水平,轉(zhuǎn)染重組質(zhì)粒pCMV-tag 2B-syncytin基因組syncytin mRNA水平比轉(zhuǎn)染空載質(zhì)粒pCMV-tag 2B組顯著增高(P0.01),表明重組質(zhì)粒pCMV-tag 2B-syncytin能成功轉(zhuǎn)染入人宮頸癌細胞中并有效表達。 結(jié)論成功構(gòu)建了syncytin基因的真核表達質(zhì)粒pCMV-tag 2B-syncytin,為進一步探討syncytin的異常表達對運動神經(jīng)元的可能損傷作用及機制奠定堅實基礎(chǔ)。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R744;R346

【共引文獻】

相關(guān)博士學(xué)位論文 前1條

1 梁巧儀;三個新的人內(nèi)源性逆轉(zhuǎn)錄病毒（HERV）相關(guān)基因的研究[D];浙江大學(xué);2009年

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本文編號：1493881