【摘要】： HIV-1慢病毒載體可感染非分裂細(xì)胞,具有轉(zhuǎn)移外源基因片段大、使目的基因長(zhǎng)期穩(wěn)定表達(dá)、不易誘發(fā)宿主免疫反應(yīng)等優(yōu)點(diǎn),是腦梗死基因治療的理想載體。本實(shí)驗(yàn)在慢病毒載體三質(zhì)粒包裝系統(tǒng)基礎(chǔ)上,將野生型HIV-1基因組分裝在四個(gè)質(zhì)粒中,構(gòu)建優(yōu)化新型HIV-1慢病毒載體四質(zhì)粒包裝系統(tǒng):載體質(zhì)粒(含指示基因——綠色熒光蛋白基因)、gag-pol質(zhì)粒(包裝質(zhì)粒)、rev質(zhì)粒和包膜質(zhì)粒,經(jīng)限制性酶切、電泳,證實(shí)質(zhì)粒構(gòu)建成功。然后經(jīng)轉(zhuǎn)化感受態(tài)細(xì)胞、搖菌,大量提取質(zhì)粒,以備轉(zhuǎn)染和測(cè)定載體滴度使用。 將四質(zhì)粒包裝系統(tǒng)共轉(zhuǎn)染293T包裝細(xì)胞,48小時(shí)后熒光顯微鏡觀察可見綠色熒光,證實(shí)質(zhì)粒轉(zhuǎn)入細(xì)胞。經(jīng)收集細(xì)胞上清及高速離心獲得HIV-1慢病毒載體顆粒。病毒載體再次轉(zhuǎn)染293T細(xì)胞,仍可觀察到綠色熒光,說明載體攜帶的指示基因成功融合入細(xì)胞基因組。經(jīng)測(cè)定病毒滴度為4×108TU/ml,這說明假包膜VSV-G使載體顆粒的穩(wěn)定性提高,通過高速離心可獲得較高滴度的病毒顆粒。優(yōu)化新型HIV-1慢病毒載體構(gòu)建成功。四質(zhì)粒系統(tǒng)的使用進(jìn)一步減少了各質(zhì)粒間的同源性,減少了重組產(chǎn)生有復(fù)制能力病毒的機(jī)會(huì)。
[Abstract]:HIV-1 lentivirus vector can infect non-mitotic cells and has the advantages of large transfer of foreign gene fragments, stable expression of the target gene for a long time, and is not easy to induce host immune response. It is an ideal vector for gene therapy of cerebral infarction. In this experiment, the wild type HIV-1 genome was divided into four plasmids on the basis of lentivirus vector three plasmid packaging system. A novel HIV-1 lentivirus vector four-plasmid packaging system was constructed: vector plasmid (containing indicator gene-green fluorescent protein gene), gag-pol plasmid (packaging plasmid), rev plasmid and envelope plasmid, restriction enzyme digestion, electrophoresis, It was confirmed that the plasmid was constructed successfully. After transformation of receptive cells, shaking bacteria, a large number of plasmid was extracted for transfection and determination of vector titer. The four plasmid packaging system was co-transfected into 293T packaging cells. 48 hours later, green fluorescence was observed by fluorescence microscope, which confirmed that the plasmid was transferred into the cells. HIV-1 lentivirus vector particles were obtained by collecting cell culture and high speed centrifugation. Green fluorescence could still be observed when 293T cells were re-transfected with virus vector, which indicated that the indicator gene carried by the vector was successfully integrated into the cell genome. The titer of the virus was 4 脳 10 ~ 8TU / ml, which indicated that the stability of the carrier particles was improved by pseudocapsule VSV-G, and the virus particles with high titer could be obtained by high speed centrifugation. The construction of a novel HIV-1 lentivirus vector was optimized successfully. The use of the four plasmid system further reduced the homology between the plasmids and reduced the chance of recombination to produce replicative viruses.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R346

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