本文關(guān)鍵詞：HIV-1整合酶抑制劑的篩選及其活性的測(cè)定　出處：《浙江大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HIV-1 整合酶 抑制劑 多肽 噬菌體展示

【摘要】： 病毒DNA整合進(jìn)宿主DNA的過程是某些反轉(zhuǎn)錄病毒在宿主細(xì)胞中增殖的關(guān)鍵步驟。由于在正常人類細(xì)胞中不存在相似的功能蛋白，其抑制劑對(duì)人體的副作用可能很小。相對(duì)于經(jīng)典AIDS治療藥物的眾多毒副作用，整合酶抑制劑理論上要具有優(yōu)勢(shì)，因此成為受人矚目的治療艾滋病的新靶點(diǎn)。以重組HIV-1整合酶為目標(biāo)蛋白，采用噬菌體展示技術(shù)，在隨機(jī)線性七肽庫(kù)中篩選與整合酶有特異結(jié)合作用的噬菌體展示肽，得到13個(gè)具有特異結(jié)合能力的陽性克��；經(jīng)DNA測(cè)序推斷出七條氨基酸序列。選取同源性較高的TPSHSSR和HPERATL兩條肽，它們可以競(jìng)爭(zhēng)性地抑制展示相應(yīng)肽段的噬菌體與整合酶的結(jié)合。通過體外整合酶活性實(shí)驗(yàn)顯示，它們對(duì)整合酶的整合活性(3’加工和鏈轉(zhuǎn)移過程)有抑制作用，TPSHSSR和HPERATL的半數(shù)抑制率即IC_(50)分別為54.56±5.18μM和28.292±1.32μM。同時(shí)，實(shí)驗(yàn)證明這兩條多肽還有抑制去整合的作用。我們的研究表明：用噬菌體展示技術(shù)可以篩選到有效的HIV-1整合酶的抑制劑，可用于整合酶機(jī)理的研究，并有潛在的新藥開發(fā)前景。 1．HIV-1整合酶的表達(dá)與純化及活性鑒定 將轉(zhuǎn)化有質(zhì)粒PT7-7-His-TX-WT-IN的E.coli BL21(DE3)菌接種于含有Amp(終濃度為100μg／ml)LB培養(yǎng)基中培養(yǎng)，加IPTG誘導(dǎo)表達(dá)后6 000 rpm，4℃離心收集菌體。超聲破碎后，15 000 rpm，4℃離心收集上清。上清經(jīng)Ni—NTA柱分離純化，得到較純的整合酶。整合酶活性的鑒定分別檢測(cè)了整合酶的整合與去整合兩個(gè)作用。整合作用采用的是一種非放射性的類似于ELISA的方法，而去整合活性則是將整合酶與相應(yīng)底物反應(yīng)，經(jīng)電泳分析底物的變化來定性鑒別。 2．噬菌體肽庫(kù)的篩選及陽性克隆的鑒定 本實(shí)驗(yàn)篩選的肽庫(kù)為隨機(jī)線性七肽庫(kù)。將純化得到的具有活性的HIV-1整合酶蛋白包被在酶標(biāo)板上，經(jīng)5％BSA封閉，噬菌體的結(jié)合，酸洗脫、中和，測(cè)滴度、擴(kuò)增等基本步驟的循環(huán)篩選后，噬菌體得到了富集，程度達(dá)到了700倍左右。將第五輪洗脫下來的噬菌體感染E.coli ER2738，挑取平板中的清晰菌斑，制備單克隆噬菌體溶液。在96孔板上包被整合酶蛋白，同時(shí)包被BSA作為陰性對(duì)照。經(jīng)封閉后加入單克隆噬菌體溶液。洗滌各孔后加入辣根過氧化物酶標(biāo)記的M13抗體，而后用TMB進(jìn)行顯色。比較各個(gè)克隆與整合酶和BSA的結(jié)合效果，篩選出特異結(jié)合整合酶蛋白而非BSA的陽性克隆。本實(shí)驗(yàn)篩選得到了13個(gè)陽性克隆。 3．DNA測(cè)序及多肽序列的推斷 提取陽性克隆的單鏈DNA，送交上海生工進(jìn)行測(cè)序，測(cè)序以—96通用引物(5'-GCCCTCATAGTTAGCGTAACG-3’)為引物。13個(gè)陽性克隆共得到7條序列。根據(jù)DNA測(cè)序結(jié)果，推測(cè)出這七條序列的氨基酸序列，分析他們之間的氨基酸組成特點(diǎn)。其中有兩條多肽的前三個(gè)氨基酸一致，另兩條多肽的第1、2和第4個(gè)氨基酸一致，，并且七條肽序列中，大部分都含有組氨酸，精氨酸，脯氨酸和絲氨酸。 4．合成七肽的競(jìng)爭(zhēng)抑制ELISA實(shí)驗(yàn) 兩條合成的七肽與展示相應(yīng)肽段的噬菌體競(jìng)爭(zhēng)性地結(jié)合整合酶的能力反映了這兩條肽的相對(duì)親和力。在96孔免疫吸附板中包被整合酶蛋白，加入不同濃度的合成多肽及一定量展示相應(yīng)肽序列的噬菌體溶液，使兩者競(jìng)爭(zhēng)結(jié)合整合酶。通過辣根過氧化物酶標(biāo)記的抗M13抗體結(jié)合，TMB顯色后，計(jì)算出噬菌體結(jié)合率：結(jié)合率％=[1-(A_(450)-A’_(450))／A_(450)]×100％；其中A_(450)為未加抑制劑時(shí)450nm波長(zhǎng)下的吸光度，A’_(450)為加入抑制劑后450 nm波長(zhǎng)下的吸光度。隨著合成多肽濃度的增加，與整合酶結(jié)合的展示相應(yīng)肽序列的噬菌體逐漸減少，結(jié)合率下降。這些結(jié)果充分證明了篩選出來的兩條多肽與整合酶蛋白特異性結(jié)合的特點(diǎn)，親和力較好。 5．多肽對(duì)整合酶活性抑制實(shí)驗(yàn) 針對(duì)整合酶的整合與去整合作用，用合成的多肽分別對(duì)這兩個(gè)反應(yīng)的抑制作用做了分析。實(shí)驗(yàn)方法與整合酶活性檢測(cè)相似，只是整合酶需要和合成的多肽先進(jìn)行預(yù)反應(yīng)。實(shí)驗(yàn)發(fā)現(xiàn)合成的兩條多肽TPSHSSR和HPERATL對(duì)整合酶的整合、去整合作用都有抑制作用。整合酶的去整合作用對(duì)整合酶的序列及結(jié)構(gòu)完整性的要求不高，只要有核心結(jié)構(gòu)域存在即可；而整合作用必須要求整合酶既有核心結(jié)構(gòu)域，又有N末端和C末端結(jié)構(gòu)域。所以，這兩條多肽對(duì)整合酶的作用位點(diǎn)很可能就在核心結(jié)構(gòu)域上。
[Abstract]:The integration of viral DNA into the host DNA process is the key step of some retroviruses in human cells. The function of protein similar does not exist in normal human cells, the side effects of inhibitors on human may be small. Compared to the side effects of classic AIDS drugs, integrase inhibitors have theory the advantage, therefore become a new target for treatment of AIDS attention. The recombinant HIV-1 integrase for target protein using phage display technology, in linear random seven peptide library screening specific binding phage display peptide and integrase, 13 with specific binding ability of positive clones by DNA; sequencing seven deduced amino acid sequence. Selected homologous TPSHSSR and HPERATL two peptide, which can competitively inhibit phage display peptide and the corresponding integrase by. In vitro experiments showed the integration of enzyme activity, enzyme activity of integration integration (3 'processing and chain transfer process) are inhibited, and the inhibition rate of half of the TPSHSSR HPERATL IC_ (50) were 54.56 + 5.18 and 28.292 + M 1.32 M. at the same time, effect of the experiment shows that the two polypeptides have inhibition to integration. Our research shows that: display technology can be screened effective inhibitors of HIV-1 integrase with phage integrase can be used to study the mechanism, and new drug development potential.
Expression and purification of 1.HIV-1 integrase and identification of its activity
The transformation of plasmid PT7-7-His-TX-WT-IN E.coli BL21 (DE3) in bacteria containing Amp (final concentration of 100 g / ml) LB medium, and after the induction of IPTG, 6000 rpm, 4 C centrifugal collection cell. After ultrasonication, 15000 rpm, 4 DEG C collected by centrifugation the supernatant by Ni - NTA. Column separation, integration of pure enzyme. Identification of integrase activity were detected by enzyme and integration to integrate two. Integration uses a non radioactive method similar to ELISA, and to integrate the activity is to integrate with the corresponding reaction enzyme substrate, by electrophoresis substrate change analysis to qualitative identification.
Screening of 2. phage peptide library and identification of positive clones
The peptide library screening experiments for linear random seven peptide library. The purified active HIV-1 integrase protein was coated on the ELISA plate, the 5%BSA closed, binding phage, acid washing, neutralization, titration, amplification and other basic steps of the cycle after screening, phage enriched, reached the level of about 700 times. The fifth round of the eluted phage infection of E.coli ER2738, were clear plaque in the plate, the preparation of monoclonal phage integrase protein coated solution. In the 96 hole plate, and coated by BSA as negative control. After being closed after adding single clone phage solution. M13 antibody of each hole after washing horseradish peroxidase labeled, then use the TMB color. Comparing the combined effect of cloning and integrase and BSA, screened the specific binding integrase protein and non BSA positive clones. This experiment screened 13 gram positive Long.
3.DNA sequencing and peptide sequence inference
The extraction of single stranded DNA positive clones, sent to Shanghai SANGON sequencing, sequencing with 96 universal primers (5'-GCCCTCATAGTTAGCGTAACG-3) primers and.13 clones were obtained 7 sequences. According to the results of DNA sequencing, deduced amino acid sequences of the seven sequences, analysis of amino acid composition between them. There are three characteristics two amino acid polypeptide, another two polypeptide 1,2 and fourth amino acids, and seven peptide sequences, most of them contain histidine, arginine, proline and serine.
Competitive inhibition ELISA experiment of 4. synthetic seven peptide
Two of seven synthetic peptides and peptide phage display corresponding competitive binding ability of integrase reflects the relative affinity of these two peptides. Coated integrase protein in 96 hole immune adsorption plate, and a certain amount of synthetic peptides with different concentrations of phage display peptide sequences of the corresponding solution. The competitive binding of integrase. By combining horseradish peroxidase labeled anti M13 antibody, TMB staining, calculate the phage binding rate: binding rate%=[1- (A_ (450) -A (450) _) / A_ (450)] * 100%; A_ (450) for the absorbance at 450nm under with the inhibitor, A (450) _ absorbance wavelength of 450 nm after adding inhibitors. With the increase of the concentration of synthetic peptides, show the corresponding peptide sequence combined with phage integrase gradually reduced, the binding rate of two. These results demonstrate that the polypeptides were screened out with the whole The specific binding of the synthase protein is good, and the affinity is good.
Inhibition of 5. polypeptide on the activity of integrase
According to the integration and integration of the role of the enzyme, with synthetic peptides of the two reaction inhibition were analyzed. Test method and integration of enzyme activity is similar, just need integrase and synthetic peptide pre reaction. The experimental results showed that the synthesis of two polypeptides TPSHSSR and HPERATL integration of the integrase to integrate, have inhibitory effect. Sequence and structural integrity to integration of the role of the integrase enzyme on the integration of the requirements is not high, as long as the core domain can; and integration must require the integration of both the core enzyme domain, and at the end of the N terminal and the C domain. Therefore, the two polypeptide action sites for integrase is likely in the core domain.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

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相關(guān)期刊論文 前2條

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2 張暉,詹金彪,許林海,嚴(yán)士q,王克夷;噬菌體肽庫(kù)展示的應(yīng)用及其最新進(jìn)展[J];中國(guó)藥理學(xué)通報(bào);2003年04期

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