本文選題：慢病毒 + 載體��； 參考：《瀘州醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的:本實(shí)驗(yàn)用PCR法擴(kuò)增出的Trail基因引入慢病毒載體系統(tǒng)，生產(chǎn)Trail慢病毒顆粒并進(jìn)行病毒滴度檢測。方法:1.Trail基因的獲得：根據(jù)GenBank中大鼠Trail基因序列(NM145681.1)，設(shè)計(jì)出該基因的特異性引物Trail-Agel-F和Trail-Agel-R，并且使用Agel酶的切位點(diǎn)。用PCR法從大鼠cDNA文庫中擴(kuò)增出目的基因。2.重組質(zhì)粒的構(gòu)建：采用In-Fusion技術(shù)，將酶切回收后的PCR產(chǎn)物線性交換連接入Agel酶切的真核表達(dá)載（GV218），產(chǎn)生目的重組質(zhì)粒。連接產(chǎn)物質(zhì)粒轉(zhuǎn)化大腸桿菌DH5a感受態(tài)細(xì)胞，擴(kuò)增目的重組質(zhì)粒。3.連接產(chǎn)物的酶切鑒定：收集擴(kuò)增后的連接產(chǎn)物，酶切后經(jīng)電泳發(fā)現(xiàn)得到片段長度均與預(yù)期相符，基因測序結(jié)果亦正確，從而證明克隆Trail基因已成功。4.重組質(zhì)粒轉(zhuǎn)染293T細(xì)胞：用脂質(zhì)體Lipofeetamine2000包裹構(gòu)建的重組質(zhì)粒和輔助包裝載體，，三質(zhì)粒共同轉(zhuǎn)染293T細(xì)胞，產(chǎn)生含有表達(dá)Trail蛋白的Lentivirus病毒顆粒。5.病毒檢測：重組質(zhì)粒轉(zhuǎn)入293T細(xì)胞,熒光顯微鏡下觀察GFP的表達(dá)，行Western blot鑒定及使用Real-time定量PCR法檢測病毒滴度。結(jié)果:成功得到Trail目的基因，重組質(zhì)粒測序結(jié)果與GenBank中Trail基因序列(NM145681.1)比較，證實(shí)Trail基因序列正確；三質(zhì)粒轉(zhuǎn)染293T細(xì)胞后熒光顯微鏡觀察到綠色熒光；WesternBlot檢測觀察到58KD附近處有特征條帶，其大小和目的基因融合蛋白相吻合；孔稀釋法及實(shí)時(shí)熒光定量PCR病毒滴度測定法測定結(jié)果為2E十8TU/ml。結(jié)論:成功克隆大鼠Trail基因和成功構(gòu)建慢病毒載體系統(tǒng)并檢測其滴度。為后續(xù)把有殺滅腫瘤的基因（Trail）轉(zhuǎn)導(dǎo)進(jìn)神經(jīng)干細(xì)胞奠定基礎(chǔ)。
[Abstract]:Aim: the Trail gene amplified by PCR method was introduced into the lentivirus vector system to produce Trail lentivirus particles and to detect the virus titer. Methods: 1. Obtaining the gene of Trail: according to the sequence of rat Trail gene NM145681.1 in GenBank, we designed the specific primers Trail-Agel-F and Trail-Agel-Rand used the restriction site of Agel. The target gene. 2. 2 was amplified from rat cDNA library by PCR. Construction of recombinant plasmid: by using In-Fusion technique, the recovered PCR products were linearly exchanged into the eukaryotic expression of Agel digested with GV218G, and the target recombinant plasmid was produced. The recombinant plasmid was transformed into Escherichia coli DH5a competent cells and the target recombinant plasmid was amplified. Identification of ligation products by enzyme digestion: the ligation products were collected and amplified. The results of electrophoresis showed that the length of the ligand fragments were in accordance with the expected results, and the sequencing results were correct, which proved that the cloned Trail gene had been successfully cloned. 4. The recombinant plasmid was transfected into 293T cells: the recombinant plasmid and the auxiliary package vector were constructed by liposome Lipofeetamine2000. The three plasmids were co-transfected into 293T cells to produce Lentivirus virus particles. 5. Virus detection: the recombinant plasmid was transferred into 293T cells. The expression of GFP was observed under fluorescence microscope. Western blot identification and Real-time quantitative PCR assay were used to detect the titer of the virus. Results: the target gene of Trail was successfully obtained, and the sequence of the recombinant plasmid was compared with the sequence of Trail gene in GenBank (NM145681.1), which confirmed that the sequence of Trail gene was correct. After the transfection of the three plasmids into 293T cells, the Western blot analysis showed that there were characteristic bands near 58KD, the size of which was consistent with the target gene fusion protein. The results of pore dilution method and real-time quantitative PCR titer assay were 2e + 8TU / ml. Conclusion: the rat Trail gene was cloned and the lentivirus vector system was successfully constructed and its titer was detected. To lay the foundation for the subsequent transduction of tumor killing gene Trail into neural stem cells.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392-33

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