本文選題：膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子 + 基因序列��； 參考：《山東大學(xué)》2005年碩士論文

【摘要】：目的:克隆人膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子(GDNF)全長基因,與連接有IRES2-EGFP的逆轉(zhuǎn)錄病毒載體pLXSN相連,構(gòu)建逆轉(zhuǎn)錄病毒真核細(xì)胞表達(dá)載體,為治療帕金森氏病、腦血管病等神經(jīng)系統(tǒng)疾病奠定基礎(chǔ)。 方法:①從人腦膠質(zhì)細(xì)胞瘤組織中提取總RNA,參照分子克隆指南,合成cDNA第1、2鏈,加入引物PCR擴(kuò)增目的基因,得到558bp的目的片段后,克隆到pMD18-T載體中,得到重組pMD18T-GDNF。②同樣方法擴(kuò)增IERS2-EGFP基因片段,克隆到pMD18-T載體中,得到重組pMD18T-IRES2-EGFP。③分別對pMD18T-GDNF,pMD18T-IRES2-EGFP,pLXSN逆轉(zhuǎn)錄病毒載體進(jìn)行酶切、連接,構(gòu)建表達(dá)質(zhì)粒。④對重組質(zhì)粒進(jìn)行酶切鑒定并測序驗(yàn)證。⑤將測序后的逆轉(zhuǎn)錄病毒重組質(zhì)粒以電穿孔的方法,轉(zhuǎn)入包裝細(xì)胞PT67表達(dá),流式細(xì)胞儀及熒光顯微鏡下觀察轉(zhuǎn)染效率。 結(jié)果:提取的總RNA反轉(zhuǎn)錄cDNA后,PCR擴(kuò)增的GDNF目的基因?yàn)?00～600bp,IRES2—EGFP基因片段,PCR擴(kuò)增的目的基因?yàn)?200～1500bp,經(jīng)酶切鑒定和序列鑒定分析,分別得到558bp和1308bp的序列,與Genebank做對照,發(fā)現(xiàn)IRES2—EGFP的片段上IRES2序列上有一個(gè)堿基突變,單它不會(huì)影響到后期熒光蛋白表達(dá)。GDNF與Genebank上發(fā)布的636bp條帶相比,缺失了78個(gè)堿基,參考相關(guān)文獻(xiàn),此缺失78堿基的基因片段仍保留原蛋白的功能,是蛋白選擇性剪切的結(jié)果,并且進(jìn)一步測序發(fā)現(xiàn),在此基因片段前蛋白的序列上出現(xiàn)了一個(gè)堿基突變,經(jīng)過查證,屬于同義突變,故不影響蛋白質(zhì)的表達(dá),表明GDNF基因和IRES2-EGFP基因克隆成功,酶切鑒定結(jié)果與預(yù)期設(shè)想一致。電轉(zhuǎn)染PT67細(xì)胞后,經(jīng)綠色熒光顯微鏡觀察和流式細(xì)胞儀測定發(fā)現(xiàn),轉(zhuǎn)染后PT67細(xì)胞在熒光顯微鏡下經(jīng)藍(lán)光激發(fā),可以觀察到大量的PT67細(xì)胞胞漿內(nèi)呈現(xiàn)綠色熒光,同時(shí)做流式細(xì)胞儀測定轉(zhuǎn)染效率,結(jié)果約為24.4%,提示EGFP基因及GDNF基因的進(jìn)一步表達(dá),說明目的基因片段已成功轉(zhuǎn)入包裝細(xì)胞內(nèi)。 結(jié)論:①利用分子生物學(xué)技術(shù),完整地克隆了人GDNF基因全序列,通過測序加以驗(yàn)證,表明該片段符合人完整的GDNF基因片段序列并將人GDNF基因連接于含IRES2-EGFP
[Abstract]:Objective: to clone the full length gene of human glial cell derived neurotrophic factor (GDNF) and to construct a retroviral eukaryotic expression vector, which is connected with the retroviral vector pLXSN connected with IRES2-EGFP, and lay the foundation for the treatment of Parkinson's disease and cerebrovascular disease.
Methods: (1) the total RNA was extracted from the human glioblastoma tissue. According to the molecular cloning guide, cDNA 1,2 chain was synthesized and the target gene was amplified by primer PCR. After the target fragment of 558bp was obtained, the recombinant pMD18T-GDNF. was cloned and the IERS2-EGFP gene fragment was amplified by the same method, and the recombinant pM was cloned to obtain the recombinant pM. The recombinant pM was cloned and the recombinant pM was obtained. The recombinant pM was cloned and the recombinant pM was obtained. D18T-IRES2-EGFP. (3) pMD18T-GDNF, pMD18T-IRES2-EGFP, pLXSN retrovirus vectors were cut, connected, and the expression plasmids were constructed. (4) the recombinant plasmid was identified by enzyme digestion and sequenced. 5. The recombinant retrovirus recombinant plasmid was sequenced by electroporation, and transferred into the PT67 expression of the packaged cell, flow cytometry and fluorescence display. The transfection efficiency was observed under microscopically.
Results: after the total RNA was back transcriptional cDNA, the GDNF target gene of PCR amplification was 500 to 600bp, IRES2 EGFP gene fragment, and the target gene of PCR amplification was 1200 ~ 1500bp. The sequence of 558bp and 1308bp was obtained by enzyme digestion and sequence identification. The base mutation, which alone does not affect the late fluorescent protein expression.GDNF and the 636bp strip published on the Genebank, is missing 78 bases, referring to the related literature. The deletion of the 78 base gene fragment still retained the function of the protein, which was the result of the protein selective shear, and further sequencing found the sequence of the preprotein in this gene fragment. A base mutation was found, which was found to be a synonymous mutation, so it did not affect the expression of protein, indicating that the GDNF gene and the IRES2-EGFP gene were cloned successfully. The results of the enzyme digestion were consistent with the expected assumption. After electrotransfection of PT67 cells, the fluorescence microscopy and flow cytometry showed that the transfected PT67 cells were fluorescent after transfection. Under the microscope, a large number of PT67 cells were observed to show green fluorescence in the cytoplasm. At the same time, the transfection efficiency was measured by flow cytometry. The result was about 24.4%, suggesting the further expression of EGFP gene and GDNF gene, indicating that the target gene fragment had been successfully transferred into the packed cell.
Conclusion: (1) the whole sequence of human GDNF gene was cloned completely by molecular biology technology, and verified by sequencing, which showed that the fragment conformed to the complete sequence of GDNF gene fragment and connected human GDNF gene to IRES2-EGFP
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 曲栗;基于逆轉(zhuǎn)錄病毒載體的表達(dá)克隆法的建立[D];東北農(nóng)業(yè)大學(xué);2010年

，

本文編號(hào)：1923089