CN117843766B - 高亲和力的抗鸡传染性法氏囊病毒的scFv抗体的制备及其应用 - Google Patents
高亲和力的抗鸡传染性法氏囊病毒的scFv抗体的制备及其应用 Download PDFInfo
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Abstract
本发明公开了高亲和力的抗鸡传染性法氏囊病毒的scFv抗体的制备及其应用。本发明提供的scFv抗体，包括由重链可变区、轻链可变区以及它们之间的连接区组成；本发明利用抗原‑抗体共表达的细菌展示技术筛选获得了8株scFv抗体，并且与IBDV VP2蛋白和不同的IBDV病毒株具有高亲和力，其可以通过构建突变库筛选出具有高亲和力和中和活性的scFv抗体，用于防治IBD；其也可以用于ELISA法检测IBDV病原。
Description
技术领域
本发明涉及高亲和力的抗鸡传染性法氏囊病毒的scFv抗体的制备及其应用，属于生物技术领域。
背景技术
鸡传染性法氏囊病(Infectious Bursal Disease，IBD)是由鸡传染性法氏囊病毒(Infectious Bursal Disease Virus，IBDV)引起的一种急性、高度接触性传染病，IBDV主要侵害3-12周龄的雏鸡和青年鸡，损伤鸡的中枢免疫器官——法氏囊。具有传播速度快、传染性强、感染率及死亡率均高的特点。该病目前在全球范围内均有分布，是养禽业最重要疾病之一，并且因免疫失败而造成的经济损失巨大。
IBDV可在雏鸡法氏囊中的淋巴细胞，尤其是B淋巴细胞中迅速繁殖，导致免疫抑制,从而可增强机体对其它病原体的易感性并且降低对其它疫苗的反应性。抗体药物目前是有效的治疗药物，高免血清和卵黄抗体在发病早期均可起到良好的效果，但是由于工业化生产可控性差和存在水平传播疾病等原因而受到限制。本发明利用抗原-抗体共表达的细菌展示技术筛选获得了8株具有较高亲和力的抗IBDV的scFv抗体，其可以通过构建突变库筛选出具有高亲和力和中和活性的scFv抗体，用于防治IBD；其也可以用于双抗夹心ELISA法检测病原IBDV的存在。
发明内容
本发明的目的是提供高亲和力的抗鸡传染性法氏囊病毒的scFv抗体及其应用。scFv抗体是由重链可变区、轻链可变区以及它们之间的连接区组成，它们能够与IBDV VP2蛋白高亲和力结合，在治疗或者预防传染性法氏囊病和检测传染性法氏囊病毒的ELISA试剂盒中具有重要应用。
附图说明
图1为scFv抗体的SDS-PAGE图谱。
图2为ELISA检测scFv抗体对VP2蛋白的特异性和亲和力的结果。
图3为ELISA检测scFv抗体对不同IBDV病毒的特异性和亲和力的结果。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
pET-27b(+)载体：购自Novagen公司，Cat.No.69337-3。大肠杆菌Rosetta：购自Novagen公司，Cat.No.71403-4。鸡传染性法氏囊病活疫苗(Gt株)：哈兽研维科生物公司，编号080012122。鸡传染性法氏囊病中等毒力活疫苗(NF8株)：扬州威克生物工程有限公司，编号101042056。鸡传染性法氏囊病活疫苗(MB株)：ABIC，编号20621150B。鸡传染性法氏囊病活疫苗(B87株)：湖南省中岸生物药业有限公司，编号180022026。
包被液(pH9.6)：取Na2CO3 0.15g、NaHCO3 0.293g，溶于水并用水定容至100mL。
PBS缓冲液：取NaCl 8g、KCl 0.2g、Na2HPO4.12H2O 3.58g、KH2PO4 0.24g，溶于1L水。
实施例1、scFv抗体及其编码基因的获得
1、抗体库的构建
取IBDV免疫后的鸡脾脏，提取RNA后反转录成cDNA。根据GenBank上的抗体序列，设计出克隆抗体可变区的基因引物，以cDNA为模板，使用PCR的方法克隆抗体可变区基因。将VH与VL片段连接成scFv基因并与细菌展示载体pBFD-Ab-VP2进行连接，构建抗IBDV抗原抗体共表达的细菌表面展示文库。VH约380bp、VL约320bp，VH-Tlinker-VL约740bp。
2、抗体库的筛选
收集转化后全部克隆，经IPTG(0.25mmol/L)诱导4h，经EDTA-MgCl2处理，与兔抗VP2蛋白的多克隆抗体(即实施例4制备的多克隆抗体溶液，工作浓度为1:5 000倍稀释)及FITC标记的羊抗兔二抗(购自sigma公司，工作浓度为1:500倍稀释)各孵育1h，PBS洗涤后利用流式细胞仪对其进行筛选。
经三轮筛选后得到8个与VP2蛋白具有高结合能力的scFv单克隆抗体，将其分别命名为scFv-11、scFv-12、scFv-13、scFv-14、scFv-15、scFv-16、scFv-17和scFv-18抗体。
8种scFv抗体的氨基酸序列如序列表的序列1～8所示，其核苷酸序列如序列表的序列9～16所示。
实施例2、8种scFv抗体的制备
1、以实施例1中筛选获得的能够表达8个具有高结合能力的单克隆抗体的质粒为模板，用F1和R1组成的引物分别对8个scFv抗体基因进行PCR扩增，得到PCR扩增产物。
F1：5'–CGCCATATGGCCGTGACGTTGGACGAG-3'；
R1：5'–CCCAAGCTTTTAACCTAGGACGGTCAGGG-3'。
2、用限制性内切酶Nde I和Hind III双酶切步骤1的PCR扩增产物，回收酶切产物。
3、用限制性内切酶Nde I和Hind III双酶切pET-27b(+)载体，回收约5367bp的载体骨架。
4、将步骤2的酶切产物和步骤3的载体骨架连接，得到重组质粒。根据测序结果，对重组质粒进行结构描述如下：在pET-27b(+)载体的Nde I和Hind III酶切位点之间插入了序列表的序列9～16所示的双链DNA分子。
5、将步骤4得到的重组质粒导入大肠杆菌Rosetta，得到重组菌。
6、将步骤5得到的重组菌接种至含50μg/ml卡那霉素的LB液体培养基，37℃、100r/min振荡培养至OD600nm＝0.3；加入IPTG并使其浓度为0.25mmol/L，37℃、100r/min振荡培养4h。
7、取15L完成步骤6的培养体系，4℃、4000r/min离心30min并收集菌体沉淀。
8、取步骤7得到的菌体沉淀，用PBS缓冲液重悬，加入溶菌酶溶液(购自Amresco)并使溶菌酶的浓度为1mg/ml，4℃放置1h，然后进行超声破碎(25瓦的功率，3min)，4℃、10000g离心30min，收集沉淀。
9、使用AKTA Purifier 100蛋白层析系统(购自GE公司)纯化目的蛋白
取步骤8得到的沉淀，用100毫升溶解缓冲液(8mol/L尿素水溶液，pH8.0)充分溶解，然后上样于HiLoad 16/60Superdex75 pg柱子(购自GE公司)，然后用500mL复性缓冲液(2mol/L尿素水溶液，pH8.0)洗脱并收集过柱后的洗脱液，然后在PBS缓冲液中透析过夜，得到溶液即为8个scFv抗体溶液。所有步骤均在4℃环境下。8种scFv抗体溶液的SDS-PAGE图谱见图1，显示约为28kD的条带，与预期相符。
实施例3、ELISA检测scFv抗体的亲和力和特异性
一、ELISA检测几种scFv抗体对VP2蛋白的特异性和亲和力
1、用蛋白浓度为10μg/ml的scFv抗体溶液(即实施例2制备的scFv抗体溶液，用包被液调整蛋白浓度)包被酶标板，4℃过夜，然后用PBST缓冲液洗涤3次，每次2min。
2、每孔加入100μl蛋白浓度为40μg/ml的VP2蛋白溶液，37℃孵育1h，然后用PBST缓冲液洗涤3次，每次2min。
3、加入兔抗VP2多克隆抗体，37℃孵育1h，然后用PBST缓冲液洗涤3次，每次2min。
4、加入HRP标记的羊抗兔抗体(购自R&D system公司，工作浓度为1:8 000倍稀释)，37℃孵育1h，然后用PBST缓冲液洗涤3次，每次2min。
5、加入TMB底物显色液，37℃避光显色5min。
6、每孔加入50μl 2mol/L的H2SO4水溶液，用酶标仪于波长450nm下检测OD值。
设置用等体积的PBS缓冲液代替步骤2中的VP2蛋白溶液和步骤3中的兔抗VP2多克隆抗体的PBS组。步骤1中用蛋白浓度为10μg/ml的scFv抗体溶液包被酶标板时：设置步骤2中不加入VP2蛋白溶液的对照组1，步骤3中不加入兔抗VP2多克隆抗体的对照组2，步骤2中不加入VP2蛋白溶液且步骤3中不加入兔抗VP2多克隆抗体的对照组3，用等体积且等蛋白浓度的BSA溶液代替VP2蛋白溶液的对照组4。
每个处理设置3个复孔。
结果见图2。ELISA结果表明，8种scFv抗体均可以与VP2蛋白特异性结合，具有高亲和力。
二、ELISA检测scFv抗体对不同IBDV病毒的特异性和亲和力
1、用蛋白浓度为10μg/ml的scFv抗体溶液(即实施例2制备的scFv抗体溶液，用包被液调整蛋白浓度)包被酶标板，4℃过夜，然后用PBST缓冲液洗涤3次，每次2min。
2、每孔加入100μl IBDV病毒液(病毒剂量为106.2TCID50)，37℃孵育1h，然后用PBST缓冲液洗涤3次，每次2min。
3、加入兔抗VP2多克隆抗体，37℃孵育1h，然后用PBST缓冲液洗涤3次，每次2min。
4、加入HRP标记的羊抗兔抗体(购自R&D system公司，工作浓度为1:8 000倍稀释)，37℃孵育1h，然后用PBST缓冲液洗涤3次，每次2min。
5、加入TMB底物显色液，37℃避光显色5min。
6、每孔加入50μl 2mol/L的H2SO4水溶液，用酶标仪于波长450nm下检测OD值。
分别采用如下IBDV的毒株进行上述实验：Gt株、NF8株、MB株、B87株。
设置用等体积的PBS缓冲液代替步骤2中的IBDV病毒液和步骤3中的兔抗VP2多克隆抗体的PBS组。设置步骤2中不加入IBDV病毒液的对照组1，步骤3中不加入兔抗VP2多克隆抗体的对照组2，步骤2中不加入IBDV病毒液且步骤3中不加入兔抗VP2多克隆抗体的对照组3，用等体积等滴度的新城疫病毒液代替IBDV病毒液的对照组4。
每个处理设置3个复孔。
结果见图3。ELISA结果表明，8种scFv抗体均可以与不同IBDV毒株特异性结合，对不同的IBDV毒株具有不同的亲和力。
Claims (5)
1.一种抗鸡传染性法氏囊病毒的单链抗体scFv-15，单链抗体scFv-15由重链可变区、轻链可变区以及它们之间的连接区组成；
所述单链抗体scFv-15为如下（a）：（a）由序列表中序列5自N末端第1-245位氨基酸残基组成的蛋白质。
2.编码权利要求1所述单链抗体scFv-15的基因。
3.如权利要求2所述的基因，其特征在于：
编码所述单链抗体scFv-15的DNA分子为如下（1）：（1）序列表的序列13自5’末端第1-738位核苷酸所示的DNA分子。
4.含有权利要求2至3中任一所述基因的表达盒、重组载体、转基因细胞系或重组菌。
5.根据权利要求1所述的单链抗体scFv-15，权利要求2或3所述的基因，或权利要求4所述的表达盒、重组载体、转基因细胞系或重组菌在制备产品中的应用；所述产品的功能为如下（Ⅰ）或（Ⅱ）或（Ⅲ）：（Ⅰ）检测鸡传染性法氏囊病病毒；（Ⅱ）辅助鉴定鸡传染性法氏囊病病毒；（Ⅲ）预防和/或治疗鸡传染性法氏囊病。
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