【摘要】： 目的構(gòu)建人的極低密度脂蛋白受體(VLDLR)基因,將其包裝成含VLDLR基因的重組腺相關(guān)病毒(rAAV-VLDLR),并檢測(cè)其滴度。方法通過(guò)設(shè)計(jì)含有特定酶切位點(diǎn)的引物,從人的骨骼肌中提取RNA,合成cDNA后,擴(kuò)增人VLDLR全長(zhǎng)基因的5’端約1000bp的DNA,并以含人VLDLR基因部分長(zhǎng)的質(zhì)粒為模板,合成人VLDLR全長(zhǎng)基因近3’端約1600bp的DNA,通過(guò)粘端連接將兩片段連接成全長(zhǎng)VLDLR,并送基因公司檢測(cè)其與文獻(xiàn)報(bào)道的全長(zhǎng)人VLDLR相符情況;將構(gòu)建的人VLDLR基因與重組腺相關(guān)病毒的真核表達(dá)載體PXXUF1連接,形成人VLDLR的重組腺相關(guān)病毒真核表達(dá)載體的重組體PXXUF1-VLDLR,鑒定后,將重組腺相關(guān)病毒系統(tǒng)的三種質(zhì)粒Pxx2、Phelper、PXXUF1-GFP(和PXXUF1-VLDLR)大量提取后純化,以293細(xì)胞作為細(xì)胞載體,利用磷酸鈣共轉(zhuǎn)染法包裝rAAV-VLDLR病毒與rAAV-GFP病毒,以rAAV-GFP病毒的包裝情況作為rAAV-VLDLR病毒包裝效率的參照,用斑點(diǎn)雜交的方法檢測(cè)兩種病毒的滴度。結(jié)果1、人極低密度脂蛋白受體基因構(gòu)建成功,與文獻(xiàn)報(bào)道基相符;2、PXXUF1-VLDLR構(gòu)建成功,rAAV-VLDLR病毒與rAAV-GFP病毒的滴度均為4×1011pfu/ml。結(jié)論用所構(gòu)建的人VLDLR基因包裝的rAAV-VLDLR病毒達(dá)到了要求的滴度,可用于后續(xù)糖尿病大鼠和細(xì)胞的相關(guān)研究。
[Abstract]:Objective To construct human very low density lipoprotein receptor (VLDLR) gene and package it into a recombinant adeno-associated virus (rAAV-VLDLR) containing VLDLR gene and detect its titer.Methods RNA was extracted from human skeletal muscle by designing primers containing specific enzyme digestion sites.After synthesizing the cDNA, the 5'-terminal DNA of the full-length gene of human VLDLR was amplified and the titer of the recombinant adeno-associated virus (rAAV-VLDLR) was determined. The human VLDLR gene was synthesized with a partial length plasmid containing human VLDLR gene as a template. The two fragments were ligated into a full length VLDLR by stick-end ligation and sent to the gene company to detect their conformity with the reported full-length human VLDLR gene. The constructed human VLDLR gene was compared with the recombinant adeno-associated virus eukaryotic expression vector PXXUF1. The recombinant adeno-associated virus eukaryotic expression vector PXXUF1-VLDLR was linked to form human VLDLR. After identification, three recombinant adeno-associated virus system plasmids Pxx2, Phelper, PXXUF1-GFP (and PXXUF1-VLDLR) were extracted and purified. 293 cells were used as cell carriers to package rAAV-VLDLR virus and rAAV-GFP disease by calcium phosphate co-transfection. Results 1. Human VLDLR gene was successfully constructed, which was consistent with the reported base. 2. PXXUF1-VLDLR was successfully constructed, and the titers of rAAV-VLDLR virus and rAAV-GFP virus were 4 *1011 pfu. Conclusion The rAAV-VLDLR virus packaged with the constructed human VLDLR gene has reached the required titer and can be used in the follow-up study of diabetic rats and cells.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R587.1;R346

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