CN106632670B - 一种猪源性抗猪传染性胃肠炎病毒的单链抗体及其制备方法 - Google Patents
一种猪源性抗猪传染性胃肠炎病毒的单链抗体及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种猪源性抗猪传染性胃肠炎病毒的单链抗体及其制备方法。该抗猪传染性胃肠炎病毒的单链抗体由抗体的重链可变区和轻链可变区通过短的连接肽连接而成。轻链可变区的氨基酸序列如SEQ ID No.1所示，重链可变区的氨基酸序列如SEQ ID No.2所示。获得的抗猪传染性胃肠炎病毒的单链抗体分子量约为28kDa，能够特异性的识别猪传染性胃肠炎病毒，可进一步用于阻断猪传染性胃肠炎病毒感染和入侵。
Description
技术领域
本发明涉及一种猪源性抗猪传染性胃肠炎病毒单链抗体及其制备方法，属于基因工程技术领域。
背景技术
猪传染性胃肠炎(Transmissible gastroenteritis，TGE)是由冠状病毒科冠状病毒属猪传染性胃肠炎病毒(TGEV)引起的急性、高度接触性传染性传染病，猪感染TGEV主要临床症状为呕吐、水样腹泻和脱水。TGE首先在1946年首次由Dolye和Hutching在美国报道，随后在欧洲、美洲、亚洲等多个国家相继报道发生病例，现在已经成为一种全世界性猪的疾病。同时TGE发病率高，各年龄段的猪均可发病，尤其是两周龄内仔猪，死亡率高达100％。PED的频发给我国养猪业造成严重的经济损失。疫苗接种是预防本病发生的途径之一，但是已研制出的PED灭活和弱毒疫苗效果并不理想。单链抗体是一种基因工程抗体，以其分子量小、特异性高、穿透力强、易于改造等特点，显示了巨大的应用潜力，越来越受到人们的重视。
发明内容
本发明的目的之一是提供一种猪源性抗猪传染性胃肠炎病毒的基因工程单链抗体，该单链抗体能够与猪传染性胃肠炎病毒特异性结合，可用于阻断猪传染性胃肠炎病毒的感染和入侵。
本发明提供的一种猪源性抗猪传染性胃肠炎病毒的单链抗体，所述单链抗体具有如SEQ ID No.1所示的重链可变区的氨基酸序列、如SEQ ID No.2所示的轻链可变区氨基酸序列和位于重链可变区和轻链可变区之间的连接肽；所述连接肽为(GGGGSGGGGSGGGGS)。
上述单链抗体具有如SEQ ID No.3所示的氨基酸序列。
本发明的另一目的是提供一种编码上述猪源性抗猪传染性胃肠炎病毒的单链抗体的基因，其具有SEQ ID No.4所示的核苷酸序列。
为了方便地对单链抗体筛选和表达纯化，在上述序列的基础上进一步设计酶切位点和识别序列，进一步含有内切酶位点SfiI和NotI识别序列，SfiI的序列为GGCCCAGCCGGCC，NotI的序列为GCGGCCGC。
本发明还提供一种表达上述猪源性抗猪传染性胃肠炎病毒的单链抗体的表达载体，所述载体为原核表达载体。优选的，所述载体为pCANTAB-5e-ScFv载体。
本发明的再一目的是提供一种制备上述猪源性抗猪传染性胃肠炎病毒的单链抗体的方法，具体步骤如下：
(1)采用RT-PCR法直接从猪传染性胃肠炎病毒感染的猪外周血RNA中扩增出抗体编码基因的重链可变区基因和轻链可变区基因；
(2)利用SOE-PCR法将连接肽与重链可变区基因和轻链可变区基因相连构建猪源性单链抗体基因；
(3)将步骤(2)的猪源性单链抗体基因克隆到噬菌粒载体中，构建重组噬菌粒；
(4)将步骤(3)的原核表达载体转化入E.coli TG1感受态细胞(北京普如汀生物技术有限公司)，培养、建立噬菌体单链抗体库；
(5)将步骤(4)表达的单链抗体用猪传染性胃肠炎病毒作为包被抗原，进行富集淘选，阳性克隆即为抗猪传染性胃肠炎病毒单链抗体的噬菌体。
(6)将步骤(5)所得阳性单链抗体噬菌体，即获得所述猪源性抗猪传染性胃肠炎病毒的单链抗体。
本发明的技术原理是采用RT-PCR直接从猪传染性胃肠炎病毒感染的猪外周血RNA中扩增出抗体编码基因的重链可变区(VH)基因和轻链可变区(VL)基因。利用SOE-PCR(重组链延伸反应)法将linker与VH基因和VL基因相连构建猪源性单链抗体(ScFv)基因，并将其克隆到噬菌粒载体pCANTAB5e中，phage-ELISA筛选抗ETEC单链抗体的阳性克隆，测序后使用DNAstar的MegAlin进行序列分析，证明该单链抗体属于猪源性抗猪传染性胃肠炎病毒的单链抗体。
和现有技术相比，本发明的有益效果是：抗猪传染性胃肠炎病毒的基因工程抗体能与猪传染性胃肠炎病毒特异性结合，能够用于猪传染性胃肠炎病的预防和治疗。
附图说明
图1为VH-Linker-VL PCR电泳图；M为2000bp DNA ladder marker；1、2为VH-Linker-VL基因PCR产物。
图2为pCANTAB5e-ScFv双酶切鉴定；M为2000bp DNA ladder marker；1、2为重组质粒经Sfi I和Not I双酶切。
图3为重组原核表达载体pCANTAB5e-ScFv质粒图谱。
图4为噬菌体抗体库的多样性分析。
图5为抗传染性胃肠炎病毒单链抗体的纯化电泳图；M为预染蛋白Marker；1为纯化后的单链抗体。
图6为单链抗体对猪传染性胃肠炎病毒的体外中和效果。
具体实施方式
实施例中各步骤采用的实验材料均为正规公司获得的标准材料,所用方法均为标准试剂盒产品说明书所述方法(见相应的实施例),各步骤获得的中间产品及最后的终产品均经过多次试验证明可以重复获得,其生物学性质保持稳定一致。说明本发明各试验步骤所涉及的中间产品和终产品均可以按照本发明所陈述的发法准确获得。
实施例1猪源性抗传染性胃肠炎病毒的单链抗体的制备
1：对养殖场出现腹泻症状的仔猪进行猪传染性胃肠炎病毒抗原检测(阳性抗原系本实验室保存的TGEV毒株)，用常规(参照F.M.奥斯伯等《精编分子生物学实验指南》)ELISA法检测血清抗体效价大于1:20000时，采集该猪血液，裂解红细胞后获得白细胞(红细胞裂解液上海碧云天生物技术有限公司)，用Trizol法(TRIZOL Reagent购自美国Invitrogen公司)提取总RNA。以提取的总RNA为模版，采用Oligo primer，根据反转录试剂盒(cDNA第1链合成试剂盒购自TaKaRa公司)的产品说明操作步骤，合成第1链cDNA。
2：对GenBank已公布的猪抗体编码基因重链可变区序列(AF064686.1；AF064687.1；AF064688.1；AF064689.1；AF064690.1)和轻链可变区序列(KF561242.1；GQ867594.1；GQ867595.1)设计扩增抗体轻、重链的引物(表1)，其中VH1F分别与VH1R、VH2R用于扩增VH区；VL1F、VL2F、VL3F和VL1R用于扩增VL区；VH3F、VH3R用于VH基因加入酶切位点和Linker序列；VL4F、VL5F、VL6F在VL1F、VL2F、VL3F扩增的VL基因基础上加入酶切位点，VL2R用于VL基因加入Linker序列。其中，VH3F含有Sfi I酶切位点，VL 2R含有Not I酶切位点；VH3R、VL4F、VL5F、VL6F含互补的Linker序列(酶切位点和Linker序列在表1中用下划线标示出)。Linker采用(GGGGS)3，其对应的编码核苷酸序列为：GGTGGCGGTGGCTCGGGCGGTGGTGGATCCGGTGGCGGCGGGTCT。引物由上海生工生物工程技术服务有限公司合成。
表1 扩增抗体可变区的引物及其扩增片段大小
注：下划线代表Sfi I或Not I酶切位点，方框代表连接肽序列。
3：VH和VL基因的扩增
以cDNA为模版，VH1F、VH1R为引物扩增VH基因。引物VL1F、VL2F、VL3F分别与引物VL1R配对，扩增VL基因。PCR反应体系为25μL：2×PCR mix12.5μL，模版cDNA 2μL，上下游引物(25μM)各1μL，ddH2O 8.5μL。扩增程序如下：95℃预变性3min；94℃变性40s，50℃退火40s，72延伸1min，30个循环；最后72℃延伸10min。1.5％琼脂糖凝胶电泳鉴定产物并回收目的基因(根据Thermo公司提供的胶回收说明书操作)。
4：ScFv基因的获得
分别以VH和VL基因为模板，VH2F、VH2R为引物PCR扩增带有Linker的重链可变区基因，VH4F、VL5F、VL 6F分别与VL2R配对，PCR扩增带有Linker的轻链可变区基因，PCR条件同上。扩增产物经1％琼脂糖凝胶电泳鉴定后回收目的基因(含有Linker序列的VH和VL基因)。以VH2 F、VL 2R为引物，通过重组链延伸反应(SOE-PCR)将含有Linker序列的VH和VL基因连接为ScFv基因，并加入Sfi I和Not I酶切位点，VH-Linker-VL扩增产物大小为714bp(见图1)。
5：猪源性ScFv噬菌体原始文库的构建
根据常规分子克隆方法(参照J.萨姆布鲁克等主编的《分子克隆实验指南》)，ScFv基因和pCANTAB-5e载体分别经Sfi I和Not I双酶切后，琼脂糖凝胶电泳并用凝胶回收试剂盒回收酶切产物(见图2)。将ScFv基因插入pCANTAB-5e载体(北京普如汀生物技术有限公司)，构建重组表达质粒(见图3)，并将其电转化E.coliTG1感受态细胞(北京普如汀生物技术有限公司)，连续电转化50次，构建猪源性ScFv噬菌体原始文库。挑取单克隆菌落PCR验证其阳性率，菌落PCR验证正确的克隆进行测序分析来确定文库的多样性(见图4)。
6：ScFv的富集筛选
取密度梯度离心获得的高浓度猪传染性胃肠炎病毒(本实验室保存)，用抗原包被液(50mmol/L碳酸氢钠盐溶液，pH＝9.6)稀释至5μg/mL，加入96孔板，每孔100μL，4℃包被过夜；每孔加入5％脱脂奶粉溶液200μL，37℃封闭1h，PBS洗涤3次；每孔加入100μL噬菌体ScFv原始文库，37℃孵育2h，PBS洗涤10次；每孔加入洗脱缓冲液(Gly-HCl pH＝2.2)100μL洗脱，再加入50μL(Tris-HCl pH＝9.0)进行中和。将部分洗脱的噬菌体感染对数生长期的大肠杆菌TG1，测定第一轮的捕获的噬菌体滴度，其余洗脱的噬菌体进行第二轮富集筛选；捕获的噬菌体滴度需要达到106cfu/mL；挑取最后一轮淘选的单菌落，即为抗猪传染性胃肠炎病毒的阳性ScFv菌落。
7：ScFv的诱导表达
挑取阳性ScFv菌落至含氨苄抗生素(终浓度为100μM)和葡萄糖(终浓度为(2M)的2YT液体培养基中，37℃振摇培养，菌液OD600至0.6时感染辅助噬菌体M13KO7(北京普如汀生物技术有限公司)。12h后离心，吸取上清，即为阳性ScFv的噬菌体。阳性ScFv的噬菌体感染对数生长期的HB2151菌液(北京普如汀生物技术有限公司)，培养至菌液OD600为0.6。将菌液分为两份，分别为诱导组、和非诱导组。诱导组在菌液中加入IPTG(终浓度100μM)，于30℃诱导过夜，离心收集菌液。用PBS悬浮菌液，超声波裂解，离心后收集上清。采用Ni-NTA hisband Rasin纯化ScFv，纯化过程参照默克公司的《pET System Manual》。纯化后的样品进行SDS-PAGE电泳(见图5)。
实施例2抗原特异性ScFv的间接ELISA筛选
取超速离心浓缩的猪传染性胃肠炎病毒(本实验室保存)，用抗原包被液(50mmol/L碳酸氢钠盐溶液，pH＝9.6)稀释至5μg/mL，加入96孔板，每孔100μL，4包被过夜；每孔加入5％脱脂奶粉溶液200μL，37封闭1h，PBS洗涤3次；将纯化蛋白的上清50μL与4％脱脂奶粉溶液50μL混匀后加入上述孔中，37℃孵育2h，PBST洗涤3次；加入E-Tag Mouse mAb(E-tag标签鼠单克隆抗体购自RayBiotech公司)100μL(1:2000)，37℃反应2h，PBST洗涤3次；加入过氧化氢标记的羊抗鼠IgG二抗(购自美国Invitrogen公司)1000μL(1:4000)，37℃反应1h，PBST洗涤3次；TMB显色15min，2mol/L硫酸终止反应，酶标仪(美国Thermo公司读取OD450处吸光值，将设未诱导菌液上清为表达阴性对照。以P/N(P为阳性孔的OD450值，N为阴性孔的OD450值)表示，P/N≥2.1为阳性；1.5≤P/N＜2.1为可疑；P/N＜1.5为阴性。阳性克隆经3次重复性试验验证，同时设猪传染性胃肠炎病毒、猪沙门菌、猪产肠毒素大肠杆菌作为对照，验证ScFv的特异性。结果证明单链抗体A能够特异性的识别猪传染性胃肠炎病毒，但不与猪流行性腹泻病毒、猪沙门菌、猪产肠毒素大肠杆菌发生交叉免疫反应。
实施例3
对获得的单链抗体编码基因进行测序，证明其由747个核苷酸及据此推测的249个氨基酸组成，所述核苷酸序列如SEQ ID No.4所示，所述氨基酸序列如SEQ ID No.3所示。
实施例4
检测单链抗体对猪传染性胃肠炎病毒的中和活性分析(见表1)。首先采用Reed-Muench方法检测TGEV感染PK15细胞的TCID50。TCID50的测定在96孔中进行，将5×105个/孔Vero细胞接种到96孔细胞培养板，37℃、5％CO2细胞培养箱中培养至细胞密度为80％；在1.5mL Eppendorf管中将病毒液分别从10-1到10-10作连续10倍梯度稀释；以无血清培养基洗涤细胞3次后将稀释好的病毒液分别入96孔板中，每一个稀释梯度接种一个纵排，每孔加入100μL病毒稀释液，最后一孔加入空白培养基做为阴性对照；37℃培养1h后，每孔补加一滴含10％胎牛血清的DMEM培养基；放置于37℃细胞培养箱中培养，逐日观察细胞病变情况，结果按照Reed-Muench法计算。公式如下：
距离比例＝(高于50％病变率的百分数-50％)/(高于50％病变率的百分数-低于50％病变率的百分数)
lgTCID50＝距离比例×稀释度对数之间的差+高于50％病变率的稀释度的对数
然后测定单链抗体的体外中和活性，分为三组。单链抗体处理组，将100μL纯化的单链抗体(100ng/μL)与100μL 1MOI病毒混合，预先作用30min，然后感染细胞；无关单链抗体处理组(证实不与TGEV结合的单链抗体)，将100μL纯化的所述单链抗体(100ng/μL)与100μL 1MOI病毒混合，预先作用30min，然后感染细胞；病毒处理组，将100μL PBS与1MOI病毒混合，预先作用30min，然后感染细胞；每组三个重复，分别在感染后6、12、18、24、30、36h收集细胞上清，测定病毒滴度，分析单链抗体的中和效果(见图6)。从结果可以看出，TGEV感染后18-36h，单链抗体处理组的病毒滴度显著低于PBS和阴性ScFv处理组(P<0.05)，说明ScFv具有中和病毒的活性。统计方法使用Student’s T test。
Claims (6)
1.一种猪源性抗猪传染性胃肠炎病毒的单链抗体，其特征在于，所述单链抗体具有如SEQ ID No.1所示重链可变区的氨基酸序列、如SEQ ID No.2所示的轻链可变区的氨基酸序列和位于重链可变区VH和轻链可变区VL之间的连接肽。
2.如权利要求1所述的单链抗体，其特征在于，其具有如SEQ ID No.4所示的氨基酸序列。
3.一种编码如权利要求1所述的单链抗体的基因，其特征在于，其具有如SEQ ID No.3所示的核苷酸序列。
4.如权利要求3所述的基因，其特征在于，所述核苷酸序列中进一步包含内切酶位点SfiI、NotI，其中SfiI为GGCCNNNNNGGCC，NotI为GCGGCCGC。
5.一种表达如权利要求1所述的单链抗体的表达载体，其特征在于，所述载体为原核表达载体。
6.如权利要求5所述的表达载体，其特征在于，所述载体为pCANTAB-5e-ScFv载体。
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