中国图书资料分类号：392.11

Inhibition of enzymatic activities of HIV-1 integrase(IN)

by the ScFv against IN

Zhang Jianhui, Li Nan, Song Xiaoguo, Meng Li, Han Baoguang, Ling Shigan, Ma Xiankai.

(Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850)

Keywords： HIVintegrasebiological activityScFv

Abstract：From bacteria strain HB2151 expressing ScFv against HIV-1 integrase (IN), three clones were selected and the soluble ScFvs were expressed and purified for testing their effects on biological activities of IN. The results showed that the 3′ end processing, strand transfer and disintegration activities of IN could be abolished by all of the three ScFvs at a ScFv/IN molar ratio of 8∶ 1, whereas BSA and antihuman cross linked fibrin ScFv did not affect any of the enzymatic activities at the same molar concentration. The three strains had the same ScFv sequence which was conformed with the DNA sequence of the variable region of mouse immunoglobulin. It was indicated that the ScFv concentration needed to abolish the 3′ end processing and disintegration reaction was higher than that of strand transfer activity. The study suggested that the in vitro enzymatic activities of IN could be inhibited by the ScFv. This result provided a good basis for further study of the possibility and mechanism of the ScFv in the replication of HIV in cells.

由于HIV基因极易突变，造成AIDS患者很容易对HIV蛋白酶、逆转录酶等抑制剂产生耐药性。近年来采用的“鸡尾酒”疗法延缓了耐药性的产生，但保持疗效的关键在于不断寻找疾病治疗的新靶位及新途径^〔1〕。

HIV整合酶(integrase,IN)为HIVpol基因3′端编码的蛋白质，可介导HIVDNA与宿主细胞染色体DNA的整合过程。由于人体内无该酶的类似物存在，IN成为AIDS治疗的理想靶位^〔2〕。为探索艾滋病治疗的新方法，我们曾利用噬菌体抗体库技术，制备了IN的特异性单链抗体(single chain fragment variable,ScFv)^〔3〕，并在大肠杆菌中进行了ScFv的可溶性表达，又以免疫亲和层析技术进行了ScFv的纯化^〔4〕。本文进一步观察了纯化的ScFv对IN蛋白体外生物学活性的影响。

1材料和方法

1.1主要材料①IN蛋白及其单链抗体：重组HIV-1 IN蛋白及鼠源性抗IN的ScFv，由本实验室研制。②同位素：γ-³²PATP为中国端辉同位素公司产品。③其它试剂及试剂盒：所用化学试剂均为分子生物纯或分析纯等级。其中T₄多核苷酸激酶购自Promega公司；G-25葡聚糖快速离心柱(G-25Sephadex Quick Spin Column)和DNA测序试剂盒均购自Pharmacia公司。④寡核苷酸：模拟的HIV-1基因长末端重复(long terminal repeats,LTR)的U5末端序列及宿主细胞DNA序列的寡核苷酸^〔6〕，由上海生工生物工程公司合成，具体序列为：

AE118：5′GTG TGG AAA ATC TCT AGC AGT 3′

AE117：5′ACT GCT AGA GAT TTT CCA CAC 3′

AE118S：5′GTG TGG AAA ATC TCT AGC A3′

AE157：5′GAA AGC GAC CGC GCC 3′

AE146：5′GGA CGC CAT AGC CCC GGC GCG GTC GCT TTC 3′

AE156：5′GTG TGG AAA ATC TCT AGC AGG GGC TAT GGC GTC C3′

1.2ScFv的可溶性表达与纯化在具有IN结合能力的可溶性ScFv中，挑选3个用ELISA法检测A值较高者，取其HB2151表达菌(S16，S18，S27)，以1L体积进行扩大培养及IPTG诱导。将每个克隆的周质腔萃取物及经硫酸铵沉淀的培养基上清合并，分别以Anti E Tag亲和层析柱(Pharmacia)纯化^〔4〕。收集纯化产物，以蔗糖浓缩至原体积的4～6倍，经IN反应缓冲液(20 mmol/L HEPES,pH7.5;10 mmol/LDTT,0.05％N-P40)充分透析，以紫外分光光度计进行蛋白定量，－20℃保存。

1.3寡核苷酸的5′端末端标记以20μl反应体系进行寡核苷酸的5′末端标记，其中含有：20pmol/L寡核苷酸50μCi〔γ-³²P〕，2μl10×T₄PNKbuffer,10μT₄PNK，混匀后，以37℃水浴1h，至70℃加热10min，终止标记反应。

1.4寡核苷酸的退火及纯化进行3′端加工反应及链转移(整合)反应时，分别于同位素标记的AE118及AE118S微量离心管中加入等量的AE117；进行去整合反应时，于标记的AE157管中，分别加入60pmol/L的AE117，AE146及A156。将以上反应管中的物质混匀后置100℃5min，逐渐降至室温，上样于G-25SephadexQuickSpincolumn,去除未标记的寡核苷酸，即得到用于酶促反应的底物。

1.5ScFv对IN体外生物活性的影响除寡核苷酸外，检测IN3种生物学活性的反应条件一致。先将抗IN蛋白的ScFv、无关蛋白(BSA)及无关ScFv(抗人交联纤维的ScFv)与纯化的IN蛋白(约30pmol/L)按一定摩尔浓度比混合，于15μl体积内(含有HEPES 20 mmol/L,pH7.5，DTT 10 mmol/L，NP-400.05％)，25℃作用30min，分别加入相应的退火、纯化后的寡核苷酸2μl，并将反应体积调节至20μl，使反应液中各成分的终浓度为：HEPES 20 mmol/L,pH 7.5,MnCl₂ 15mmol/L,NaCl 50 mmol/L(来自IN复性液)，DTT 10 mmol/LNP-40 0.05％。混匀后置37℃水浴1h，立即加入20μl上样缓冲液(98％去离子甲酰胺，10 mmol/L EDTA,pH8.0,0.05％溴酚蓝)终止反应。

1.6变性聚丙烯酰胺凝胶电泳及放射自显影将酶促反应管以100℃加热5min后，上样(10 μl)于含有7 mol/L尿素的19％聚丙烯酰胺凝胶中，30mA下电泳4h，揭胶后进行放射自显影。

1.7ScFvDNA的序列测定将以上3株ScFv的HB2151菌提取质粒，以Sanger双脱氧核苷酸末端终止法，按试剂盒操作说明进行ScFv DNA的序列分析。

2结果

2.1IN蛋白的生物学活性图1A(见第I页)为IN蛋白的3′端加工活性示意图。寡核苷酸AE118与AE117退火，可模拟HIV-1线性DNALTR序列的U5末端。IN蛋白可特异性地识别该末端，将其正链(AE118)3′端的终末两碱基切除，形成一3′端凹陷的结构。将21bp的AE118 5′端以同位素标记，经切除两碱基后可显示19bp的反应产物(图2A，见第I页)。IN蛋白也可以退火的AE118和AE117作为宿主细胞的靶DNA，将其随机切割，使具有3′端凹陷的HIV-1U5末端可随机整合入内，此即为IN的链转移活性或称为整合活性。寡核苷酸AE118S可代表已切除两碱基的HIV-1基因U5末端。将其5′端进行同位素标记且与AE117退火后，可直接作为IN链转移活性的反应供体，并可以另一对寡核苷酸为受体，进行链转移(整合)反应(图1B，见第I页)。经PAGE电泳显示，在底物上方有数条呈梯状的整合产物存在(图2A，2B，见第I页)。

图13′端加工(A)、链转移(B)及去整合活性(C)（示意图）

Fig1Diagram showing 3′-end processing (A), strand transfer

(B) and disintegration (C) of IN

Open circles denote the 5′-end. The location of the 5′-³²P-labeles are denoted by a filled circle. Filled triangles indicate site of cleavage. The numbers in parentheses indicate the lengths in nucleotides of the oligonucleotides or the reaction product.

图2抗IN单链抗体对3′端加工(A)、链转移(B)及去整合活性(C)的抑制作用

Fig2Inhibition of 3′-end processing (A), strand transfer

(B) and disintegration (C) by ScFv against IN

The filled arrow corresponds to the 5′-end-labeled input substrate. The open arrows indicate the positions of the products. The IN was incubated with BSA (lane3), unrelated ScFv (lane 4) and ScFv against IN (lane 5～7). The oligonucleotide substrate was incubated with IN in the absence of other proteins in lane 2.

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