核基質(zhì)附著區(qū)調(diào)控CHO細(xì)胞轉(zhuǎn)基因表達(dá)序列分子特征的研究
發(fā)布時(shí)間：2019-06-19 17:57
【摘要】：背景利用基因重組技術(shù)可生產(chǎn)具有治療作用的重組蛋白。針對重組蛋白的生產(chǎn),目前應(yīng)用最多的細(xì)胞表達(dá)系統(tǒng)是中國倉鼠卵巢(Chinese hamster ovary,CHO)細(xì)胞表達(dá)系統(tǒng)。但是CHO細(xì)胞表達(dá)系統(tǒng)存在轉(zhuǎn)基因表達(dá)不穩(wěn)定,表達(dá)水平較低等問題。前期研究發(fā)現(xiàn)核基質(zhì)附著區(qū)(matrix attachment region,MAR)能克服轉(zhuǎn)基因沉默,提高轉(zhuǎn)基因的表達(dá)水平。然而,MAR調(diào)控轉(zhuǎn)基因表達(dá)的機(jī)制、關(guān)鍵元件尚不清楚。目的分析核基質(zhì)附著區(qū)調(diào)控轉(zhuǎn)基因表達(dá)的分子序列特征,鑒定能有效提高CHO細(xì)胞轉(zhuǎn)基因表達(dá)的MAR特征性元件。方法根據(jù)NCBI GeneBank中人β-珠蛋白MAR序列,將人β-珠蛋白MAR全長共分為大小幾乎相等的6段進(jìn)行PCR克隆。設(shè)計(jì)PCR引物,引物的兩端引入Kpn I和Bgl II酶切位點(diǎn)。以人全血基因組為模板,利用PCR技術(shù)依次從β-珠蛋白MAR的5′和3′端分段漸次克隆MAR各片段,片段之間相互重疊120 bp。將克隆好的MAR片段進(jìn)行Kpn I和Bgl II雙酶切,連接到載體pCATG啟動(dòng)子的上游,轉(zhuǎn)化大腸桿菌DH5α感受態(tài)細(xì)菌,構(gòu)建重組質(zhì)粒pCAM1-6。將重組質(zhì)粒pCAM1-6載體、pCAM和pCATG空載體轉(zhuǎn)染CHO細(xì)胞,G418加壓選擇篩選,獲得穩(wěn)定轉(zhuǎn)染CHO細(xì)胞克隆株,用ELISA方法檢測不同穩(wěn)定轉(zhuǎn)染組中氯霉素乙酰轉(zhuǎn)移酶(chloramphenicol acetyltransferase,CAT)的表達(dá)量,生物信息學(xué)分析各片段MAR序列特征。結(jié)果結(jié)果表明,β-珠蛋白MAR全長能顯著提高轉(zhuǎn)基因的表達(dá),6個(gè)漸次片段相比較,421~1 020位的第2個(gè)分段和901~1 500位的第3個(gè)分段提高轉(zhuǎn)基因表達(dá)作用最顯著。對MAR-1~MAR-6進(jìn)行生物信息學(xué)分析發(fā)現(xiàn):MAR-2含有1個(gè)MAR-like motif(AATATATTT);MAR-3含有1個(gè)MAR-like motif(AATATATTT)、2個(gè)Topo II位點(diǎn);MAR-6含有1個(gè)Alu保守序列及β-globin cluster。結(jié)論MAR元件能夠提高哺乳動(dòng)物細(xì)胞中轉(zhuǎn)基因的表達(dá);MAR元件中MAR-like motif有助于轉(zhuǎn)基因表達(dá)的提高。
[Abstract]:Background of the invention recombinant proteins having therapeutic effects can be produced using a gene recombination technique. For the production of recombinant protein, the most recently used cell expression system is Chinese Hamster Ovary (CHO) cell expression system. However, the expression system of CHO cells has the problems of unstable transgene expression and low expression level. The preliminary study found that the matrix attachment region (MAR) can overcome the transgenic silencing and improve the expression level of the transgene. However, MAR controls the mechanism of the transgene expression, and the key elements are not yet clear. Objective To study the molecular sequence characteristics of the gene expression in the nuclear matrix attachment region, and to identify the characteristic elements of MAR which can effectively improve the expression of the CHO cells. Methods According to the sequence of human-globin MAR in NCBI GeneBank, the full length of human-globin MAR was divided into 6 segments with almost the same size to carry out PCR cloning. PCR primers were designed and both ends of the primer were introduced into the Kpn I and Bgl II cleavage sites. By using the human whole blood genome as a template, the fragment of MAR was cloned from the 5-and 3-end of the MAR-globin MAR by PCR, and the fragments were overlapped with each other by 120 bp. The cloned MAR fragment was digested with Kpn I and Bgl II and ligated to the upstream of the vector pCATG promoter to transform the competent bacteria of E. coli DH5 to construct the recombinant plasmid pCAM1-6. The recombinant plasmid pCAM1-6 vector, pCAM and pCATG empty vector were transfected into CHO cells and G418 was selected for selection, and the CHO cell clones were stably transfected. The characteristics of MAR sequences were analyzed by bioinformatics. The results showed that the full length of the MAR-globin MAR could significantly improve the expression of the transgene, and the expression of the transgenic expression was most significant in the second segment of 421-1 020 and the third segment of 901-1,500. The bioinformatics analysis of MAR-1-MAR-6 found that MAR-2 contains 1 MAR-like motif (AATATTT), MAR-3 contains 1 MAR-like motif (AATATTT),2 Topo II sites, and MAR-6 contains 1 Alu conserved sequence and 1-global cluster. Conclusion MAR elements can improve the expression of transgenes in mammalian cells, and MAR-like motif in MAR elements contributes to the improvement of transgene expression.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78
本文編號(hào)：2502538
[Abstract]:Background of the invention recombinant proteins having therapeutic effects can be produced using a gene recombination technique. For the production of recombinant protein, the most recently used cell expression system is Chinese Hamster Ovary (CHO) cell expression system. However, the expression system of CHO cells has the problems of unstable transgene expression and low expression level. The preliminary study found that the matrix attachment region (MAR) can overcome the transgenic silencing and improve the expression level of the transgene. However, MAR controls the mechanism of the transgene expression, and the key elements are not yet clear. Objective To study the molecular sequence characteristics of the gene expression in the nuclear matrix attachment region, and to identify the characteristic elements of MAR which can effectively improve the expression of the CHO cells. Methods According to the sequence of human-globin MAR in NCBI GeneBank, the full length of human-globin MAR was divided into 6 segments with almost the same size to carry out PCR cloning. PCR primers were designed and both ends of the primer were introduced into the Kpn I and Bgl II cleavage sites. By using the human whole blood genome as a template, the fragment of MAR was cloned from the 5-and 3-end of the MAR-globin MAR by PCR, and the fragments were overlapped with each other by 120 bp. The cloned MAR fragment was digested with Kpn I and Bgl II and ligated to the upstream of the vector pCATG promoter to transform the competent bacteria of E. coli DH5 to construct the recombinant plasmid pCAM1-6. The recombinant plasmid pCAM1-6 vector, pCAM and pCATG empty vector were transfected into CHO cells and G418 was selected for selection, and the CHO cell clones were stably transfected. The characteristics of MAR sequences were analyzed by bioinformatics. The results showed that the full length of the MAR-globin MAR could significantly improve the expression of the transgene, and the expression of the transgenic expression was most significant in the second segment of 421-1 020 and the third segment of 901-1,500. The bioinformatics analysis of MAR-1-MAR-6 found that MAR-2 contains 1 MAR-like motif (AATATTT), MAR-3 contains 1 MAR-like motif (AATATTT),2 Topo II sites, and MAR-6 contains 1 Alu conserved sequence and 1-global cluster. Conclusion MAR elements can improve the expression of transgenes in mammalian cells, and MAR-like motif in MAR elements contributes to the improvement of transgene expression.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78
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