發(fā)布時間：2018-05-06 01:16

  本文選題：IKK2dn + 基因轉(zhuǎn)移��； 參考：《蘇州大學》2008年碩士論文

【摘要】： 目的: (1)構(gòu)建用于真核細胞表達的重組腺病毒載體pAdxsi-GFP-IKK2dn;(2)將其包裝成腺病毒顆粒,并進一步轉(zhuǎn)染哺乳細胞hela并驗證其在細胞中表達,為進一步研究轉(zhuǎn)IKK2dn基因在同種異體器官移植中誘導免疫耐受,保護移植物的作用奠定基礎(chǔ)。 方法: (1)將pACCMVpLpASR(+)-IKK2dn基因與pShuttle-GFP-CMV(-)TEMP重組穿梭載體經(jīng)KpnI/HindIII酶切,回收和純化。T4DNA連接酶進行連接后轉(zhuǎn)化為感受態(tài)細胞DH5a,丁胺卡那霉素篩選。提取單克隆菌落,作酶切鑒定,抽取陽性菌落即得到pShuttle-GFP-CMV(-)TEMP-IKK2dn重組穿梭載體質(zhì)粒。(2)將重組穿梭載體質(zhì)粒pShuttle-GFP-CMV(-)TEMP-IKK2dn與重組腺病毒載體質(zhì)粒pAdxsi經(jīng)I-CeuI+I-SceI雙酶切處理,回收和純化。T4DNA連接酶進行連接后轉(zhuǎn)化為感受態(tài)細胞DH5a,氨芐青霉素篩選。提取單克隆菌落,作酶切鑒定,抽取陽性菌落即得到pAdxsi-GFP-IKK2dn病毒質(zhì)粒以用于病毒包裝。(3)脂質(zhì)體法將重組腺病毒載體pAdxsi-GFP-IKK2dn和Lipofectamine2000脂質(zhì)體共轉(zhuǎn)染于人胚腎細胞系的293T細胞。觀察細胞出毒跡象并進行包裝,收毒,凍融,擴增,再收毒并作毒種鑒定后以離心,透析方法純化腺病毒進行滴度測定及檢測。(4)并將腺病毒顆粒轉(zhuǎn)染哺乳細胞hela并驗證其在細胞中表達。 結(jié)果: (1)得到7.4kbp的Shuttle-GFP-CMV(-)TEMP-IKK2dn重組穿梭載體質(zhì)粒。(2)腺病毒載體pAdxsi-GFP-IKK2dn序列測定結(jié)果正確。(3)最后獲得的腺病毒載體滴度為2x1011 PFU/ml。(4) RT-PCR驗證腺病毒顆粒成功轉(zhuǎn)染哺乳細胞hela,可供進一步實驗研究需要。 結(jié)論: (1)成功構(gòu)建Shuttle-GFP-CMV(-)TEMP-IKK2dn重組穿梭載體質(zhì)粒。(2)成功構(gòu)建攜帶IKK2dn基因重組腺病毒載體pAdxsi-GFP-IKK2dn。(3)重組腺病毒載體pAdxsi-GFP-IKK2dn和Lipofectamine2000脂質(zhì)體成功包裝成高滴度腺病毒顆粒。(4)腺病毒顆粒成功轉(zhuǎn)染哺乳細胞hela,為進一步研究轉(zhuǎn)IKK2dn基因在同種異體器官移植中誘導免疫耐受,保護移植物提供了理論和實驗基礎(chǔ)。
[Abstract]:Objective: to construct a recombinant adenovirus vector pAdxsi-GFP-IKK2dn2 for eukaryotic cell expression. The recombinant adenovirus vector pAdxsi-GFP-IKK2dn-2 was packaged into adenovirus granules and transfected into lactation cells hela and verified its expression in the mammalian cells. The results provide a basis for further study on the role of IKK2dn gene in inducing immune tolerance and protecting grafts in allogeneic organ transplantation. Methods: the recombinant shuttle vector pACCMVpLpASRN and pShuttle-GFP-CMV(-)TEMP was digested by KpnI/HindIII, and then purified by KpnI/HindIII. The recombinant plasmid was transformed into a receptive cell line DH 5a, and was screened with amikacin. The recombinant shuttle vector of pACCMVpLpASRN was digested by KpnI/HindIII. The recombinant shuttle vector plasmid pShuttle-GFP-CMV(-)TEMP-IKK2dn and the recombinant adenovirus vector pAdxsi were digested by I-CeuI I-SceI, and the recombinant shuttle vector pShuttle-GFP-CMV(-)TEMP-IKK2dn and the recombinant adenovirus vector pAdxsi were digested by I-CeuI I-SceI, respectively, and the recombinant shuttle vector plasmid pShuttle-GFP-CMV(-)TEMP-IKK2dn and the recombinant adenovirus vector plasmid pAdxsi were digested by I-CeuI I-SceI, and the recombinant shuttle vector plasmid pShuttle-GFP-CMV(-)TEMP-IKK2dn and recombinant adenovirus vector plasmid pAdxsi were digested by I-CeuI I-SceI. The DNA ligase of .T4 was recovered and purified, and then transformed into the competent cell line DH 5a for screening ampicillin. Monoclonal colonies were extracted and identified by enzyme digestion. PAdxsi-GFP-IKK2dn virus plasmids were extracted from the positive colonies to be used for viral packaging. The recombinant adenovirus vectors pAdxsi-GFP-IKK2dn and Lipofectamine2000 liposomes were co-transfected into 293T cells of human embryonic kidney cell line by liposome method. Observe the signs of cytotoxicity and pack it up, collect it, freeze and thaw it, amplify it, and then identify the virus species and centrifuge it. The adenovirus was purified and detected by dialysate method. The adenovirus particles were transfected into the lactation cells hela and verified its expression in the cells. Results: 1) the recombinant Shuttle-GFP-CMV(-)TEMP-IKK2dn shuttle vector plasmid of 7.4kbp was obtained. The result of pAdxsi-GFP-IKK2dn sequencing of adenovirus vector was correct. 3) finally, the titer of adenovirus vector was 2x1011 PFU / ml. 4) RT-PCR was used to verify the successful transfection of adenovirus particles into mammalian cells, which could be used for further experimental study. Conclusion: the recombinant shuttle vector plasmid of Shuttle-GFP-CMV(-)TEMP-IKK2dn was successfully constructed. The recombinant adenovirus vector pAdxsi-GFP-IKK2dn.f3) the recombinant adenovirus vectors pAdxsi-GFP-IKK2dn and Lipofectamine2000 liposomes were successfully packaged into high-titer adenovirus particles. In order to further study the induction of immune tolerance in allogeneic organ transplantation with IKK2dn gene, Hela was stained with lactation cells. Protection of grafts provides theoretical and experimental basis.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前8條

1 王娟;顧錦法;楊水云;肖田;齊榮;孫蘭英;劉新垣;;癌特異性雙靶向載體的安全性及其攜帶基因的殺傷性[J];癌癥;2006年04期

2 楊文宇,黃宗海,湯福祥,錢勇,車小燕;“兩步轉(zhuǎn)化法”高效制備攜帶自殺基因的重組腺病毒載體[J];第一軍醫(yī)大學學報;2004年02期

3 尹冰楠,李冬田,李秋香;一種簡易、廉價、高效構(gòu)建重組腺病毒載體的方法[J];天津醫(yī)科大學學報;2005年02期

4 吳艦宇,宋春芳,許評,劉銳;來源骨髓的樹突狀細胞培養(yǎng)方法特征及其合理時間[J];中國臨床康復;2005年23期

5 林濤;丁杰;孟繁平;韓全利;喻召才;郭長存;劉志國;樊代明;;利用細菌內(nèi)重組腺病毒系統(tǒng)構(gòu)建胃癌MG7-Ag模擬表位疫苗[J];世界華人消化雜志;2003年01期

6 林勇;陳偉忠;謝渭芬;張忠兵;;重組復制缺陷型腺病毒基因治療肝纖維化的研究與應(yīng)用[J];世界華人消化雜志;2003年03期

7 李U，

本文編號：1850129