CN101712719A - 人源化中和性抗流感ns1基因工程抗体制备方法与用途 - Google Patents
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Abstract
本发明涉及一种人源化抗流感病毒NS1蛋白中和性抗体的制备方法与用途。通过运用基因工程技术和噬菌体表面展示技术，直接从人抗体基因库中筛选能与甲型流感病毒NS1抗原特异性结合的抗体，获得其抗体基因并表达，用作临床抗甲型病毒药物评价和甲型病毒治疗。
Description
技术领域
本发明涉及一种人源化单克隆抗体的制备方法与用途，具体涉及一种人源化抗流感病毒NS1蛋白中和性抗体的制备方法与用途。
背景技术
甲型流感是甲型流感病毒引起的一种急性、人畜共患呼吸道传染性疾病。它的传染性极强，极易造成世界范围内的大流行。历史上流感的大流行曾经夺去许多人的生命，据世界卫生组织(WHO)发布的公告，全球每年流感病例为6亿～12亿例，死亡50万～100万人，其中重症流感病例300万～500万例，重症流感的病死率为8％～10％。2009年在墨西哥暴发并迅速在全球蔓延的人感染甲型(H1N1)流感疫情目前已造成近10万人感染，上千人死亡，引发全球恐慌。目前仍无特效的治疗流感的方法。又由于其抗原变异性强，宿主范围广，各亚型间几乎没有交叉保护性，使得流感难以控制和频繁爆发。而作为一类新型特异性抗病毒感染的生物工程制剂--中和性抗体，在抗感染治疗及紧急被动免疫预防方面一直扮演着重要的角色。因此，研制一种针对流感病毒的广谱中和效应的抗体用于紧急预防和治疗是十分必要和可行的。
甲型H1N1病毒属于正粘病毒科(Orthomyxoviridae)，甲型流感病毒属(Influenza virus A)，其遗传物质为RNA。甲型H1N1流感病毒携带有H1N1亚型猪流感病毒毒株，包含有禽流感、猪流感和人流感三种流感病毒的核糖核酸基因片断，同时拥有亚洲猪流感和非洲猪流感病毒特征。典型病毒颗粒呈球状，直径为80nm～120nm，有囊膜。囊膜上有许多放射状排列的突起糖蛋白，分别是血凝素HA、神经氨酸酶NA和M2蛋白。病毒颗粒内为核衣壳，呈螺旋状对称，直径为10nm。猪流感病毒为单股负链RNA病毒，基因组约为13.6kb，由大小不等的8个独立片段组成。共编码10个多肽，其中节段1编码PB2蛋白，节段2编码PB1蛋白，节段3编码PA蛋白，节段4编码血凝素(HA)，节段5编码核蛋白(NP)，节段6编码神经氨酸酶(NA)，节段7编码基质蛋白1和2(M1/2)，节段8编码非结构蛋白1和2(NS1/2)。在8个基因片段中最短的是NS1基因。由于HA和NA分子容易发生抗原漂移或突变(每年每核苷突变率为10-3)，其抗原性表现出很大的变异，而且A型流感病毒亚型众多，各亚型诱导的抗体不能相互交叉保护。
而NS1蛋白是流感病毒NS1基因编码的一种小分子、非结构的RNA结合蛋白，可以编码202-237氨基酸，能与特定RNA序列结合，然后对基因表达的转录后行为进行调节，是一个具有多种活性的调控因子。NS1蛋白只在病毒感染的细胞中合成，并未被包装进病毒颗粒。在病毒感染早期即有大量NS1蛋白表达，其在细胞浆合成后很快转移至核内，并积聚在病毒感染早期的胞核内，在感染后期则聚积于核仁中，形成致密的晶体样包涵体，提示NS1蛋白在病毒复制及早期抵抗机体的抗病毒免疫过程中具有重要作用。反向遗传系统实验证实了NS1蛋白在保护流感病毒抵抗细胞IFN反应中的重要作用，当缺乏NS1基因时，重组病毒A/PR/8/34(H1N1)仅能在细胞IFN反应缺陷时复制。研究表明，非结构蛋白及其抗体可以作为病毒感染机体的一个重要标记。
绝大多数甲型流感病毒NS1蛋白具有两个比较重要的功能区：A区和B区。A区为氨基端的RNA结合区，NS1蛋白可通过此区与不同种类的RNA包括dsRNA相结合；B区为分子羧基端的效应区，此区可与宿主细胞核蛋白相互作用，抑制宿主细胞核mRNA的运输。通过这两种途径，NS1蛋白可拮抗IFN-α/β，抑制细胞mRNA前体加工和mRNA转运，促进病毒mRNA翻译，增强病毒复制等。因此，拮抗NS1蛋白的功能对于抑制甲型流感病毒复制和限制病毒传播至关重要。
随着抗体库技术的出现，抗体工程进入崭新的发展阶段。通过基因重组技术将抗体分子在基因水平上重组、表达、纯化，可获得多种多样的特异性的人源化抗体，为将来可能的临床诊断、预防和治疗提供可行性的。
发明内容
本发明的目的：是通过运用基因工程技术和噬菌体表面展示技术，直接从人抗体基因库中筛选能与甲型流感病毒NS1抗原特异性结合的抗体，获得其抗体基因并表达，用作临床抗甲型病毒药物评价和甲型病毒治疗。
本发明是这样实现的：NS1蛋白只在病毒感染的细胞中合成，并未被包装进病毒颗粒。在病毒感染早期即有大量NS1蛋白表达，其在细胞浆合成后很快转移至核内，并积聚在病毒感染早期的胞核内，在感染后期则聚积于核仁中，形成致密的晶体样包涵体，提示NS1蛋白在病毒复制及早期抵抗机体的抗病毒免疫过程中具有重要作用。反向遗传系统实验证实了NS1蛋白在保护流感病毒抵抗细胞IFN反应中的重要作用，当缺乏NS1基因时，重组病毒A/PR/8/34(H1N1)仅能在细胞IFN反应缺陷时复制。本发明采用NS1蛋白及其抗体可以作为病毒感染机体的一个重要标记这一特性，通过运用基因工程技术和噬菌体表面展示技术，直接从人抗体基因库中筛选能与甲型流感病毒NS1抗原特异性结合的抗体，获得其抗体基因并表达，用作临床抗甲型病毒药物评价和甲型病毒治疗。
本发明的积极效果：
一、提供一种新型的流感病毒及其药物评价检测方法。
二、提供一种可针对幼儿和老人等不适宜接种流感疫苗的人群进行流感病毒感染后的治疗。
具体实施方案
以下的优先实施例对本发明做详细说明，但不意味着限制本发明的内容。
利用基因工程技术克隆人甲型流感病毒H1N1亚型NS1基因，在原核表达系统表达NS1蛋白，通过提取、纯化获得纯度较高的NS1蛋白。用NS1蛋白做抗原对噬菌体抗体库进行富集筛选，并在E.coli中进行分泌表达。通过ELISA、间接免疫荧光试验(IFA)鉴定抗体对人甲型H1N1流感病毒特异性结合的功能活性，并进行序列测定。然后将阳性克隆的轻链和重链Fd段基因，分别克隆入全抗体表达载体Pac-L-Fc，转染昆虫Sf9细胞，利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达。用ELISA、IFA、Western Blot和流式细胞仪对所获人源单抗的功能特性进行鉴定。
实施例1：抗原制备
利用基因工程技术将流感病毒NS1基因重组至表达载体pTXB 1转化感受态E.coliBL21(DE3)，挑取LB(Amp+)琼脂平板上的单菌落，接种于5ml LB(Amp+)培养基，37℃振荡培养过夜，次日按1∶100的比例转种，扩大培养至A600＝0.5～0.7时，加入终浓度为1.0mmol/LIPTG，37℃表达6小时用50ml Columnbuffer平衡几丁质亲和柱，取超声破菌的上清液上样，用80ml Column buffer去除杂蛋白，再用用30ml 30mmol/L DTT[使用前切割缓冲液(20mmol/LTris-HCl 500mmol/L NaCl，0.1mmol/L EDTA pH 8.0)稀释]慢速冲洗柱子，关闭出样口，4℃切割过夜。次日用不含DTT的30ml切割缓冲液冲洗，收集目的蛋白峰，将所获得的目的蛋白溶液置于透析袋内，在磁力搅拌器的作用下，以蒸馏水为透析液透析48h，用蔗糖覆盖透析袋以浓缩蛋白采用Bradford法对纯化的重组蛋白进行定量，并检测相对分子质量。
实施例2：噬菌体抗体库的生物筛选
以100g/ml的NS1抗原溶液150μl(溶于0.1mol/L pH 8.6的NaHCO3)包被96孔酶标板，4℃孵育过夜。弃包被液，加满1％BSA封阻液，4℃作用2h。用TBS缓冲液快速洗板6次。用100μl的TBST[Tris-HCl缓冲液+0.1％(V/V)Tween-20]缓冲液稀释2×1011 pfu的噬菌体，然后加到已包被好的孔中，室温温和摇动60min。TBST缓冲液洗板10次，然后加入0.2mol/LGlycine-HCl(pH2.2)洗脱15min，再用1mmol/L Tris-HCl 15μl(pH 9.1)中和上述洗脱液，即第1轮特异性结合噬菌体。把第1轮筛选物进行扩增和纯化，然后依次进行第2次和第3次亲和筛选。每次加入的噬菌体量都为2×1011pfu，第2、3轮筛选包被的抗原量分别为10、1μg/ml，TBST中Tween-20的浓度增至0.5％(V/V)，其余步骤与第一轮筛选相同。
实施例3：特异性噬菌体的扩增和纯化
接种E.coliER2738单菌落于20ml LB培养基中，摇床培养至对数前期。加入未扩增洗脱物。37℃剧烈摇动培养4.5h。然后4℃，10000×g离心10min。取上清10000×g离心。将上清的上部80％转入一新鲜管中，加入1/6PEG/NaCl。噬菌体4℃沉淀过夜。4℃，10000×g离心15min。弃上清再短暂离心。沉淀物重悬于1ml TBS中，4℃离心5min使残余细胞沉淀。取上清，用1/6体积的PEG/NaCl再沉淀。冰上孵育60min。4℃10000×g离心10min，弃上清。沉淀物重悬于200μl TBS，0.02％NaN3中。10000×g离心1min，取上清，此即为扩增后的洗脱物。
实施例4：噬菌体滴度的测定和挑取蓝色噬菌斑
接种ER2738单菌落于10mlLB培养基中，摇床培养至对数中期。分成200μl等份于微量离心管中，每管加入10μl不同稀释度的噬菌体，快速震荡混匀，室温温育3min。将感染细胞加入45℃预温的上层琼脂培养管中，每次管，快速混匀，立即倾注于37℃预温的LB/IPTG/X-gal平板上，37℃培养过夜。次日计算噬菌体滴度。从第3轮筛选后的洗脱产物测定滴度后，用灭菌牙签在总量不到100个噬菌斑的平板上随机挑选20个蓝色噬菌斑，分别置1ml接种了处于对数生长前期的E.coli ER2738的LB培养液中进行扩增和纯化并测定滴度。
实施例5：ELISA鉴定特异性结合的阳性噬菌体克隆
用100μl 100μg/ml的NS1抗原(溶于0.1mol/LpH 8.6NaHCO3中)包被ELISA板，4℃包被过夜。弃包被液，满1％BSA封阻液，4℃作用1h。TBST洗板6次，加入TBST系列稀释的扩增噬菌体，100μl/孔。室温震荡作用1h。TBST洗板6次，加入HRP标记的抗M13抗体，200μl/孔，室温震荡作用1h。TBST洗板6次，加入TMB显色液显色，2mol/L H2SO4终止显色，测定450nm处的光密度(OD)值。用抗体库中的噬菌体作阴性对照，TBST作空白对照。
实施例6：竞争ELISA检测噬菌体阳性克隆特异性
用包被液稀释的NS1抗原(100μg/ml)包被酶标板4℃过夜，4℃封闭1h后，加50μl(1011 pfu/ml)噬菌体溶液与50μl不同浓度(50、25、12.5、6.25μg/ml)的NS1抗原混合液，37℃孵育1h，TBST[Tris-HCl缓冲液+0.5％(V/V)Tween-20)洗涤，再加入1∶5000稀释的HRP标记的抗M13抗体100μl/孔，37℃孵育1h，以TMB显色10min后，2mol/L H2SO4终止显色，酶标仪读取OD值(波长450nm)。
实施例7：使用人源化NS1抗体检测流感病毒
双抗体夹心酶免试剂的研制：将抗NS1的单克隆抗体利用CB稀释到1mg/ml，以每孔100μl的量包被到酶标板上；同时将抗NS1的单克隆抗体同辣根过氧化物酶或碱性磷酸酶相偶联，制成双抗体夹心检测试剂。将疑似患者的鼻腔分泌物、口腔分泌液、血液等稀释一倍后取100μl加到加样孔，37℃温育30分钟，洗板五次后，将NS1单克隆抗体同辣根过氧化物酶或碱性磷酸酶偶联的偶联物稀释到10μg/ml加入到加样孔中，37℃温育30分钟，洗板五次后，加入显色剂显色，如果显色即可认为该患者感染流感病毒。
金标试纸条的研制：将NS1单克隆抗体用PBS(Ph7.8，0.02M PB，0.17M NaCl)稀释到4mg/ml，以点或线的形式包被到NC膜上，同时将NS1的单克隆抗体包被到金、硒等显色物质上制成试纸条，将可疑性患者的鼻腔分泌物、口腔分泌物、血液等稀释一倍后取50μl点到加样膜上，过10-15分钟如果包被NS1单克隆抗体的包被线显色即可认为该患者感染流感病毒。
实施例8：使用人源化NS1抗体进行病毒中和实验
一定浓度的流感病毒与倍比稀释的NS1抗体等量混合，室温放置1h。取9-10日龄鸡胚，75％酒精消毒后气室朝上置于超净台，用无菌镊子、剪刀在气室端开一小口，从小口中滴入无菌的液体石蜡，轻轻晃动鸡胚，使液体石蜡在鸡胚壳膜内层铺开。注射器吸取标本刺入尿囊腔，每个鸡胚接种0.2ml。每个稀释度接种4只鸡胚，无菌医用胶布封口后37℃孵育72h至7天，每天取一只胚检查鸡胚尿囊液的血凝活性，不出现血凝活性的最高稀释度即为血清中和抗体滴度。
Claims (2)
1.人源化中和性抗流感NS1基因工程抗体制备方法与用途，其特征为：通过运用基因工程技术和噬菌体表面展示技术，直接从人抗体基因库中筛选能与甲型流感病毒NS1抗原特异性结合的抗体，获得其抗体基因并表达，用作临床抗甲型病毒药物评价和甲型病毒治疗。
2.如权利要求1所述的NS1基因工程抗体，其特征是可与流感病毒发生中和反应。
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