HIV-1整合酶随机突变库的构建

发布时间：2018-04-20 13:41

本文选题：HIV-1 + 整合酶　；参考：《浙江大学》2013年硕士论文

【摘要】：研究背景整合酶对于HIV-1病毒的复制至关重要,同时整合酶在HIV-1生命周期的脱壳与逆转录过程中均发挥了重要作用,使得整合酶成为除蛋白酶、逆转录酶以外的一个非常具有前景的抗HIV治疗的新靶点。目前已有两个经FDA批准的整合酶抑制剂上市,并且同时还有另外一些整合酶抑制剂已进入临床试验阶段。然而,有研究团队发现,一些含有整合酶突变体的病毒株对于正在进行临床实验的或者是已经上市的整合酶抑制剂具有耐受性。化学治疗能够抑制HIV-1病毒的复制以及AIDS的进一步发展,然而在此过程中具有药物耐受性的病毒突变体的出现大大降低了化学治疗的效果,对于治疗来说是一个主要的障碍。新药物的开发,以及对于具有耐受性的病毒基因型数据的分析处理,对于加强治疗效果极其重要。然而,要鉴别出导致药物耐受性的突变需要多年的临床研究。传统的体外实验在获得可靠的耐药性数据方面存在一定的局限性。为了鉴别出具有药物耐受性的整合酶突变体,我们引入了转座子介导的碱基替换突变(transposon-directed base-exchange mutagenesis, TDEM)的方法。该方法能够制造一个在目的基因上突变位点均匀分布,且每个目的基因的分子含有一至两个氨基酸随机突变的一个突变库。方法我们首先将转座子通过转座反应随机插入到整合酶基因中。转座子的两端具有识别并与转座酶结合的区域以及NotI内切酶识别位点。转座反应完成后,通过NoiI内切酶酶切去除转座子并打开转座子所在的位置,露出因NotI酶切而得到的粘性末端,并将同样用NotI酶切得到的突变插入片段(MI)与之进行连接。之后通过BsgI与BpmI两轮酶切,即可去除MI中的多余序列,保留预先设计好的三个连续碱基,这三个碱基便可实现对目的基因中连续三个碱基的替换突变。最后用pFrameCheck质粒对得到的突变体进行筛选,以排除出现的移码突变或者因突变产生的终止密码子。结果突变后未经FrameCheck过程筛选的质粒,共获得6个成功进行碱基替换的质粒,约占总数的1/3,这与文献中的结果是相似的。具体测序结果如下：共送样20个突变质粒,返回16个结果,4个测序失败。在返回的16个结果中,7个产生了理想的三个碱基的替换突变,1个完全未突变(可能由于经突变的序列与原序列完全一致),1个有一个碱基的插入,2个有大于三个碱基的插入,1个有一个碱基的缺失,1个有两个碱基的缺失,3个有大于三个碱基的缺失。在产生了三个碱基替换的7个中,1个产生了终止密码子,6个是成功进行了碱基突变。在这6个中,有2个产生突变后所编码的氨基酸与原来的一致；另外4个成功进行碱基突变并产生氨基酸突变(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).
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91;Q55

【参考文献】