本文選題：HIV-1 + 整合酶�。� 參考：《浙江大學(xué)》2013年碩士論文

【摘要】：研究背景 整合酶對于HIV-1病毒的復(fù)制至關(guān)重要,同時整合酶在HIV-1生命周期的脫殼與逆轉(zhuǎn)錄過程中均發(fā)揮了重要作用,使得整合酶成為除蛋白酶、逆轉(zhuǎn)錄酶以外的一個非常具有前景的抗HIV治療的新靶點。目前已有兩個經(jīng)FDA批準(zhǔn)的整合酶抑制劑上市,并且同時還有另外一些整合酶抑制劑已進(jìn)入臨床試驗階段。然而,有研究團(tuán)隊發(fā)現(xiàn),一些含有整合酶突變體的病毒株對于正在進(jìn)行臨床實驗的或者是已經(jīng)上市的整合酶抑制劑具有耐受性。 化學(xué)治療能夠抑制HIV-1病毒的復(fù)制以及AIDS的進(jìn)一步發(fā)展,然而在此過程中具有藥物耐受性的病毒突變體的出現(xiàn)大大降低了化學(xué)治療的效果,對于治療來說是一個主要的障礙。新藥物的開發(fā),以及對于具有耐受性的病毒基因型數(shù)據(jù)的分析處理,對于加強治療效果極其重要。然而,要鑒別出導(dǎo)致藥物耐受性的突變需要多年的臨床研究。傳統(tǒng)的體外實驗在獲得可靠的耐藥性數(shù)據(jù)方面存在一定的局限性。為了鑒別出具有藥物耐受性的整合酶突變體,我們引入了轉(zhuǎn)座子介導(dǎo)的堿基替換突變(transposon-directed base-exchange mutagenesis, TDEM)的方法。該方法能夠制造一個在目的基因上突變位點均勻分布,且每個目的基因的分子含有一至兩個氨基酸隨機(jī)突變的一個突變庫。 方法 我們首先將轉(zhuǎn)座子通過轉(zhuǎn)座反應(yīng)隨機(jī)插入到整合酶基因中。轉(zhuǎn)座子的兩端具有識別并與轉(zhuǎn)座酶結(jié)合的區(qū)域以及NotI內(nèi)切酶識別位點。轉(zhuǎn)座反應(yīng)完成后,通過NoiI內(nèi)切酶酶切去除轉(zhuǎn)座子并打開轉(zhuǎn)座子所在的位置,露出因NotI酶切而得到的粘性末端,并將同樣用NotI酶切得到的突變插入片段(MI)與之進(jìn)行連接。之后通過BsgI與BpmI兩輪酶切,即可去除MI中的多余序列,保留預(yù)先設(shè)計好的三個連續(xù)堿基,這三個堿基便可實現(xiàn)對目的基因中連續(xù)三個堿基的替換突變。最后用pFrameCheck質(zhì)粒對得到的突變體進(jìn)行篩選,以排除出現(xiàn)的移碼突變或者因突變產(chǎn)生的終止密碼子。 結(jié)果 突變后未經(jīng)FrameCheck過程篩選的質(zhì)粒,共獲得6個成功進(jìn)行堿基替換的質(zhì)粒,約占總數(shù)的1/3,這與文獻(xiàn)中的結(jié)果是相似的。具體測序結(jié)果如下：共送樣20個突變質(zhì)粒,返回16個結(jié)果,4個測序失敗。在返回的16個結(jié)果中,7個產(chǎn)生了理想的三個堿基的替換突變,1個完全未突變(可能由于經(jīng)突變的序列與原序列完全一致),1個有一個堿基的插入,2個有大于三個堿基的插入,1個有一個堿基的缺失,1個有兩個堿基的缺失,3個有大于三個堿基的缺失。在產(chǎn)生了三個堿基替換的7個中,1個產(chǎn)生了終止密碼子,6個是成功進(jìn)行了堿基突變。在這6個中,有2個產(chǎn)生突變后所編碼的氨基酸與原來的一致；另外4個成功進(jìn)行堿基突變并產(chǎn)生氨基酸突變(P142C, H171P, N254I, E270Q)。
[Abstract]:Research background Integrase is very important for replication of HIV-1 virus, and integrase plays an important role in the process of HIV-1 life cycle demudation and reverse transcription, which makes integrase become protease removal. A very promising new target for anti-HIV therapy beyond reverse transcriptase. At present, two integrase inhibitors approved by FDA have been put on the market, and some other integrase inhibitors have entered the clinical trial stage. However, the team found that some strains containing integrase mutants are resistant to integrase inhibitors that are being tested in clinical trials or are already on the market. Chemotherapy can inhibit the replication of HIV-1 virus and the further development of AIDS. However, the emergence of drug-tolerant mutants greatly reduces the efficacy of chemotherapy, which is a major obstacle to the treatment. The development of new drugs and the analysis of genotypic data of tolerant viruses are very important to enhance the therapeutic effect. However, identifying mutations that lead to drug tolerance requires many years of clinical research. Traditional in vitro experiments have some limitations in obtaining reliable data on drug resistance. In order to identify integrase mutants with drug tolerance, transposon-directed base-exchange mutagenesis (TDEM) was introduced. This method can produce a mutation library with random mutation of one or two amino acids in each target gene. Method We first randomly inserted transposons into integrase genes through transposon reactions. The two ends of transposon have recognized and bound to transposase and NotI endonuclease recognition sites. After transposing, the transposon was removed by NoiI endonuclease digestion, and the position of transposon was opened to reveal the sticky end of transposon, and the mutant insert fragment, which was also digested by NotI, was connected with the transposon. After two rounds of BsgI and BpmI digestion, the superfluous sequences in MI can be removed, and the three consecutive bases designed in advance can be retained. These three bases can realize the substitution mutation of the three consecutive bases in the target gene. Finally, pFrameCheck plasmids were used to screen the mutants to exclude the frameshift mutation or the terminating codon produced by the mutation. Result Six plasmids with successful base substitution were obtained after mutation without screening by FrameCheck process, accounting for about one third of the total, which is similar to the results in the literature. The results of sequencing were as follows: a total of 20 mutant plasmids were collected, 16 results were returned and 4 failed. Of the 16 returned results, 7 produced ideal three base substitutions, 1 completely unmutated (probably because the mutated sequence was completely consistent with the original sequence, 1 had one base insertion and 2 had more than 3 bases) In the insertion, one deletion of one base, one deletion of two bases, and three deletion of more than three bases were found. Of the 7 of the three base substitutions, 1 produced a termination codon and 6 successfully carried out base mutation. Two of the 6 mutated amino acids were identical to the original amino acids, the other 4 were successfully mutated and produced amino acid mutations (P142C, H171P, N254Iand E270QN).
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R512.91;Q55

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 朱森康;黃磊;李燕飛;鐘衛(wèi)鴻;徐志南;;制備高效大腸桿菌電轉(zhuǎn)化感受態(tài)細(xì)胞和電轉(zhuǎn)化條件的研究[J];生物技術(shù)通報;2011年10期

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