發(fā)布時(shí)間：2018-03-29 13:06

  本文選題：HIV　切入點(diǎn)：逆轉(zhuǎn)錄酶　出處：《中南大學(xué)》2013年博士論文

【摘要】：目的：含有AZT相關(guān)突變位點(diǎn)的HIV對(duì)新藥114的耐藥機(jī)制研究 方法：實(shí)驗(yàn)分為細(xì)胞內(nèi)和細(xì)胞外兩個(gè)部分。細(xì)胞內(nèi)實(shí)驗(yàn)首先通過克隆產(chǎn)生野生型和含有突變位點(diǎn)病毒的質(zhì)粒,其中突變位點(diǎn)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變位點(diǎn)的病毒,通過病毒感染含有熒光素酶的TAM-bl細(xì)胞,比較新藥114與AZT,3TC, TDF對(duì)野生型和突變型病毒的抑制作用(IC50值)的差異。 細(xì)胞外實(shí)驗(yàn)分為酶促動(dòng)力學(xué)反應(yīng)和引物延伸實(shí)驗(yàn)兩個(gè)部分。首先構(gòu)建含有的M41L/T215Y, M41L/L210W/T215Y, T69SSS, T69SSS/L210W/T215Y和T69SSS/M41L/L210W/T215Y的EPH質(zhì)粒,通過細(xì)菌擴(kuò)增產(chǎn)生大量逆轉(zhuǎn)錄酶。其次,通過NI-NTA提取法提取大量野生型和含有突變位點(diǎn)的逆轉(zhuǎn)錄RT酶,并通過FPLC法純化蛋白。第三,酶促動(dòng)力學(xué)反應(yīng),在沒有ATP存在下,比較天然底物dTTP和NRTIs衍生物AZT TP、D4T TP、114TP之間的競(jìng)爭(zhēng)性抑制作用。第四,引物延伸實(shí)驗(yàn),加入生理濃度的ATP,比較在ATP存在下,野生型RT和突變型RT催化下,結(jié)合在引物上的AZT MP、D4T MP、114MP能否都被切除,探討新藥114與AZT, D4T在M41L/T215Y,M41L/L210W/T215Y, T69SSS, T69SSS/L210W/T215Y和T69SSS/M41L/L210W/T215Y這些突變位點(diǎn)的RT作用下,是否有相同的耐藥機(jī)制。 結(jié)果： 1.細(xì)胞內(nèi)實(shí)驗(yàn)通過瓊脂凝膠電泳和基因測(cè)序結(jié)果證明,成功構(gòu)建了野生型和含有AZT相關(guān)耐藥位點(diǎn)HIV復(fù)制模型。3TC, AZT,114和TDF四種NRTIs類藥物對(duì)野生型病毒都有很強(qiáng)的抑制作用,其中,是3TC的17倍,114的82倍,TDF的42倍。多重突變M41L/L210W/T215Y和T69SSS/M41L/L210W/T215Y對(duì)3TC有較強(qiáng)的耐藥性,是野生型的20余倍；M41L/L210W/T215Y和T69SSS/M41L/L210W/T215Y對(duì)AZT耐藥最明顯,其耐藥性分別升高126和1235倍；T69SSS突變和/或者合并有M41L、L210W、T215Y多重突變可以增強(qiáng)病毒對(duì)TDF20-48倍的耐藥性；單一位點(diǎn)突變M41L和T215Y對(duì)114的耐藥性基本無影響,而M41L/T215Y, M41L/L210W/T215Y和T69SSS/M41L/L210W/T215Y,耐藥性分別升高13倍,13倍和25倍。與AZT,3TC和TDF相比,114對(duì)突變病毒的抑制作用最強(qiáng)。 2.細(xì)胞外實(shí)驗(yàn)通過基因測(cè)序和酶活性測(cè)定證明,成功構(gòu)建野生型和含有AZT相關(guān)突變位點(diǎn)的逆轉(zhuǎn)錄酶。酶促動(dòng)力學(xué)部分,RT催化D/D反應(yīng)的的活性是D/R4-8倍。酶促動(dòng)力學(xué)參數(shù)Km值,當(dāng)P/T為D/D和D/R時(shí)相比較,在D/R時(shí)Km值明顯較小。競(jìng)爭(zhēng)性抑制反應(yīng)參數(shù)Ki值,P/T為D/R情況下明顯小于P/T為D/D時(shí),這與細(xì)胞內(nèi)實(shí)驗(yàn)IC50值的結(jié)果一致。其中,114TP的Ki值最小,其次為AZT TP, D4T TP的Ki值最大,在野生型RT下,114TP與引物結(jié)合效率是AZT TP的2倍,是D4T TP的20余倍。引物延伸實(shí)驗(yàn)部分,在ATP存在下,加入AZT TP和114TP后,23bp產(chǎn)物基本沒有變化；在沒有ATP存在下,23bp產(chǎn)物基本逐漸減少。在ATP的存在下,結(jié)合在引物上的AZT MP,114MP和D4T MP在WT RT作用下,24bp產(chǎn)物基本沒有變化,而M41L/L210W/T215Y和T69SSS/M41L/L210W/T215Y突變的RT催化下,24bp產(chǎn)物減少。 結(jié)論： 1.成功建立了野生型和含有AZT相關(guān)突變位點(diǎn)的HIV復(fù)制細(xì)胞模型體系；成功構(gòu)建野生型和含有AZT相關(guān)耐藥位點(diǎn)的HIV逆轉(zhuǎn)錄重組表達(dá)質(zhì)粒。 2.細(xì)胞內(nèi)實(shí)驗(yàn)表明,3TC, AZT,114和TDF四種NRTIs類藥物對(duì)野生型病毒AZT的抑制作用最強(qiáng),對(duì)突變型病毒114的抑制作用最強(qiáng)。 3.細(xì)胞外實(shí)驗(yàn)表明,RT催化下dTTP和AZT TP, D4T TP,114TP的競(jìng)爭(zhēng)性抑制反應(yīng)主要發(fā)生在逆轉(zhuǎn)錄過程；在沒有ATP存在下,野生型與突變型RT對(duì)AZT TP,D4T TP和114TP與引物結(jié)合的效率沒有影響；在ATP存在下,突變型RT對(duì)結(jié)合在引物上的AZT MP,114MP和D4T MP的有切除作用 4.114對(duì)M41L/L210W/T215Y, T69SSS, T69SSS/L210W/T215Y和T69SSS/M41L/L210W/T215Y這些突變的耐藥機(jī)制可能產(chǎn)生ATP依賴的焦磷酸解作用,結(jié)合細(xì)胞內(nèi)實(shí)驗(yàn)結(jié)果,114對(duì)突變病毒仍有較強(qiáng)的抑制作用,提示114是潛在的抗HIV藥物
[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 .

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.91

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 姜曉華,龍亞秋;A Simple and Highly Efficient Preparation of Structurally Di-verse Aryl ?diketoacids as HIV-1 Integrase Inhibitors[J];Chinese Journal of Chemistry;2004年09期

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本文編號(hào)：1681255