HIV对逆转录酶抑制剂2’，3’-双脱氢-3’-脱氧-4’-乙炔基胸苷（114）耐药机制的研究

发布时间：2018-03-29 13:06

本文选题：HIV　切入点：逆转录酶　出处：《中南大学》2013年博士论文

【摘要】：目的：含有AZT相关突变位点的HIV对新药114的耐药机制研究方法：实验分为细胞内和细胞外两个部分。细胞内实验首先通过克隆产生野生型和含有突变位点病毒的质粒,其中突变位点包括T69SSS, M41L, T215Y, M41L/T215Y, M41L/L210W/T215Y, T69SSS/L210W/T215Y和T69SSS/M41L/L210W/T215Y。其次,制作大量野生型和含有突变位点的病毒,通过病毒感染含有荧光素酶的TAM-bl细胞,比较新药114与AZT,3TC, TDF对野生型和突变型病毒的抑制作用(IC50值)的差异。细胞外实验分为酶促动力学反应和引物延伸实验两个部分。首先构建含有的M41L/T215Y, M41L/L210W/T215Y, T69SSS, T69SSS/L210W/T215Y和T69SSS/M41L/L210W/T215Y的EPH质粒,通过细菌扩增产生大量逆转录酶。其次,通过NI-NTA提取法提取大量野生型和含有突变位点的逆转录RT酶,并通过FPLC法纯化蛋白。第三,酶促动力学反应,在没有ATP存在下,比较天然底物dTTP和NRTIs衍生物AZT TP、D4T TP、114TP之间的竞争性抑制作用。第四,引物延伸实验,加入生理浓度的ATP,比较在ATP存在下,野生型RT和突变型RT催化下,结合在引物上的AZT MP、D4T MP、114MP能否都被切除,探讨新药114与AZT, D4T在M41L/T215Y,M41L/L210W/T215Y, T69SSS, T69SSS/L210W/T215Y和T69SSS/M41L/L210W/T215Y这些突变位点的RT作用下,是否有相同的耐药机制。结果： 1.细胞内实验通过琼脂凝胶电泳和基因测序结果证明,成功构建了野生型和含有AZT相关耐药位点HIV复制模型。3TC, AZT,114和TDF四种NRTIs类药物对野生型病毒都有很强的抑制作用,其中,是3TC的17倍,114的82倍,TDF的42倍。多重突变M41L/L210W/T215Y和T69SSS/M41L/L210W/T215Y对3TC有较强的耐药性,是野生型的20余倍；M41L/L210W/T215Y和T69SSS/M41L/L210W/T215Y对AZT耐药最明显,其耐药性分别升高126和1235倍；T69SSS突变和/或者合并有M41L、L210W、T215Y多重突变可以增强病毒对TDF20-48倍的耐药性；单一位点突变M41L和T215Y对114的耐药性基本无影响,而M41L/T215Y, M41L/L210W/T215Y和T69SSS/M41L/L210W/T215Y,耐药性分别升高13倍,13倍和25倍。与AZT,3TC和TDF相比,114对突变病毒的抑制作用最强。 2.细胞外实验通过基因测序和酶活性测定证明,成功构建野生型和含有AZT相关突变位点的逆转录酶。酶促动力学部分,RT催化D/D反应的的活性是D/R4-8倍。酶促动力学参数Km值,当P/T为D/D和D/R时相比较,在D/R时Km值明显较小。竞争性抑制反应参数Ki值,P/T为D/R情况下明显小于P/T为D/D时,这与细胞内实验IC50值的结果一致。其中,114TP的Ki值最小,其次为AZT TP, D4T TP的Ki值最大,在野生型RT下,114TP与引物结合效率是AZT TP的2倍,是D4T TP的20余倍。引物延伸实验部分,在ATP存在下,加入AZT TP和114TP后,23bp产物基本没有变化；在没有ATP存在下,23bp产物基本逐渐减少。在ATP的存在下,结合在引物上的AZT MP,114MP和D4T MP在WT RT作用下,24bp产物基本没有变化,而M41L/L210W/T215Y和T69SSS/M41L/L210W/T215Y突变的RT催化下,24bp产物减少。结论： 1.成功建立了野生型和含有AZT相关突变位点的HIV复制细胞模型体系；成功构建野生型和含有AZT相关耐药位点的HIV逆转录重组表达质粒。 2.细胞内实验表明,3TC, AZT,114和TDF四种NRTIs类药物对野生型病毒AZT的抑制作用最强,对突变型病毒114的抑制作用最强。 3.细胞外实验表明,RT催化下dTTP和AZT TP, D4T TP,114TP的竞争性抑制反应主要发生在逆转录过程；在没有ATP存在下,野生型与突变型RT对AZT TP,D4T TP和114TP与引物结合的效率没有影响；在ATP存在下,突变型RT对结合在引物上的AZT MP,114MP和D4T MP的有切除作用 4.114对M41L/L210W/T215Y, T69SSS, T69SSS/L210W/T215Y和T69SSS/M41L/L210W/T215Y这些突变的耐药机制可能产生ATP依赖的焦磷酸解作用,结合细胞内实验结果,114对突变病毒仍有较强的抑制作用,提示114是潜在的抗HIV药物
[Abstract]:Objective : To study drug resistance mechanism of new drug 114 with HIV - related mutation site .

Methods : The experiments were divided into two parts : intracellular and extracellular . The intracellular experiment first produced wild - type and mutant - site - containing plasmids by cloning . The mutation sites included T69SSS , M41L , T215Y , M41L / T215Y , M41L / L210W / T215Y , T69SSS / L210W / T215Y and T69SSS / M41L / L210W / T215Y . Secondly , a large number of wild - type and mutant - containing viruses were produced .

A large number of reverse transcriptase ( RT - PCR ) was constructed by the method of NI - T69SSS / T215Y , M41L / L210W / T215Y , T69SSS , T69SSS / L210W / T215Y and T69SSS / M41L / L210W / T215Y .

Results :

1 . The results of agarose gel electrophoresis and gene sequencing showed that the wild - type and anti - HIV replication models containing the anti - HIV replication model were successfully constructed . The results showed that there were strong inhibitory effects on wild - type virus in the 3TC , HPL210W / T215Y and T69SSS / M41L / L210W / T215Y , which were more than 20 times of wild type .
The drug resistance of M41L / L210W / T215Y and T69SSS / M41L / L210W / T215Y was the most significant for the drug resistance , and the drug resistance was increased by 126 and 23times , respectively .
T69SSS mutation and / or combined M41L , L210W , T215Y multiple mutations can enhance the resistance of the virus to TDF20 - 48 ;
The resistance of M41L / T215Y , M41L / L210W / T215Y and T69SSS / M41L / L210W / T215Y increased 13 - fold , 13 - fold and 25 - fold respectively .

2 . In addition , the Km value of TP and D4T TP was the lowest when P / T was D / D and D / R . The results showed that the Ki value of 114TP was the lowest , and the binding efficiency of 114TP and D4T TP was 2 times that of D4T TP .
In the absence of ATP , the 23bp product was substantially reduced . In the presence of ATP , the 24 bp product was substantially unchanged under the action of WT RT , but the 24 bp product was reduced by RT catalyzed by M41L / L210W / T215Y and T69SSS / M41L / L210W / T215Y mutation .

Conclusion :

1 . Successfully established a model system of HIV replication cells with wild - type and mutation sites associated with the same ;
The recombinant expression plasmid of HIV reverse transcription was constructed successfully .

2 . In - cell experiments showed that the inhibitory effect of 3TC , ZAP , TDF and TDF on the wild - type virus was the strongest , and the inhibitory effect on mutant virus 114 was the strongest .

3 . Out - of - cell experiments showed that the competitive inhibition response of dTTP and the TP , D4T TP and 114TP in RT - catalyzed RT - catalyzed reverse transcription was mainly in the reverse transcription process .
In the absence of ATP , wild - type and mutant RT had no effect on the efficiency of combination with the primer combination of the TP , D4T TP , and 114TP ;
In the presence of ATP , the mutant RT has a cut - off effect on the combination of a primer - binding partner of the same

4.114 The mechanism of drug resistance of these mutations in M41L / L210W / T215Y , T69SSS , T69SSS / L210W / T215Y and T69SSS / M41L / L210W / T215Y may produce ATP dependent pyrophosphoric acid solution .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.91

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