【摘要】：目的：擴增純化鑒定重組腺病毒基因治療載體rAd-preS1，，體外多種細(xì)胞實驗檢測其對肝細(xì)胞的嗜向性。 方法：同源重組法構(gòu)建了含HBV嗜肝性基因PreS1的重組腺病毒質(zhì)粒prAd-PreS1，酶切鑒定后293細(xì)胞包裝生成腺病毒rAd-preS1。聚合酶鏈反應(yīng)PCR檢測目的基因片段Pres1。經(jīng)反復(fù)感染293細(xì)胞擴增獲得較高滴度重組腺病毒，氯化銫經(jīng)典法純化病毒。用rAd-preS1體外分別感染人正常肝細(xì)胞（L02）、人肺癌細(xì)胞(A549)、人喉癌細(xì)胞（Hep2）檢測其肝嗜向性，同時野生腺病毒rAd作為對照，觀察各細(xì)胞中熒光蛋白GFP表達(dá)情況，比較各細(xì)胞內(nèi)的病毒滴度值，同時用流式細(xì)胞術(shù)檢測各細(xì)胞重組腺病毒的感染率，并進(jìn)行統(tǒng)計學(xué)分析。 結(jié)果：酶切鑒定證實rAd-preS1載體構(gòu)建成功，擴增后病毒滴度1.15×10~9efu/ml。PCR結(jié)果示大小750bp目的基因片段。rAd-preS1轉(zhuǎn)染多種細(xì)胞示L02細(xì)胞中綠色熒光蛋白表達(dá)明顯強于A549、Hep2細(xì)胞，L02細(xì)胞的病毒滴度值高于A549、Hep2，有顯著差異(P值均0．01)，而A549、Hep2細(xì)胞間表達(dá)差異無統(tǒng)計學(xué)意義(P值0．05)；對照組各細(xì)胞間表達(dá)差異無統(tǒng)計學(xué)意義(P值0．05)。流式細(xì)胞術(shù)結(jié)果示人肝細(xì)胞感染率顯著高于非肝細(xì)胞組。 結(jié)論：攜HBV PreS1重組腺病毒載體rAd-preS1對人肝細(xì)胞相對較強嗜向性，而對非肝細(xì)胞無明顯效果，野生腺病毒無明顯細(xì)胞嗜向性，為進(jìn)一步研究肝臟疾病靶向基因治療奠定了基礎(chǔ)。
[Abstract]:Aim: to detect the tropism of recombinant adenoviral gene therapy vector rAd-preS1, to hepatocytes by amplification and purification in vitro. Methods: the recombinant adenoviral plasmid PreS1 containing HBV hepatophilic gene was constructed by homologous recombination method and identified by restriction endonuclease digestion. The adenoviral rAd-preS1. was generated in the packaging of 293cells. Detection of target gene fragment Pres1. by polymerase chain reaction PCR The recombinant adenovirus with high titer was obtained by repeated infection of 293cells, and the virus was purified by cesium chloride classical method. Human normal hepatocytes (L02), human lung cancer cells (A549) and human laryngeal cancer cells (Hep2) were infected with rAd-preS1 in vitro to detect their hepatic tropism. At the same time, wild adenovirus rAd was used as control to observe the expression of fluorescent protein GFP in each cell. The virus drop values in each cell were compared, and the infection rate of recombinant adenoviruses in each cell was detected by flow cytometry and statistically analyzed. Results: enzyme digestion confirmed that the rAd-preS1 vector was successfully constructed. The titer of the virus was 1.15 脳 10 ~ 9efu/ml.PCR. The expression of green fluorescent protein in L02 cells was significantly stronger than that in A549 cells. The results showed that the expression of green fluorescent protein in L02 cells was significantly stronger than that in A549. the expression of green fluorescent protein in L02 cells was significantly stronger than that in A549 cells. The viral drop values of Hep2 cells and L02 cells were significantly higher than those of A549 and Hep2 cells, but there was no significant difference in the expression of A549 and Hep 2 cells. There was no significant difference in the expression of each cell in the control group (P 0.05). The results of flow cytometry showed that the infection rate of human hepatocytes was significantly higher than that of non-hepatocytes. Conclusion: the recombinant adenoviral vector rAd-preS1 carrying HBV PreS1 has relatively strong tropism to human hepatocytes, but no obvious effect on non-hepatocytes, and no obvious cell tropism for wild adenoviruses, which lays a foundation for further study of targeted gene therapy for liver diseases.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R450;R512.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 譚平萍;馮德云;;非病毒性載體在肝臟疾病基因治療中的研究進(jìn)展[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2009年06期

2 李丹,王小眾,陳治新,黃月紅;乙型肝炎病毒preS1基因真核表達(dá)載體的構(gòu)建及表達(dá)[J];中西醫(yī)結(jié)合肝病雜志;2002年05期

3 牛明福;吉萍;武漢良;邵勇;高麗華;胡顯文;;preS1-preS2-HBsAg真核表達(dá)載體的構(gòu)建及在293細(xì)胞中的表達(dá)[J];中國藥理學(xué)與毒理學(xué)雜志;2012年06期

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