CN117069854A - 一种抗达氟沙星单链抗体、核酸分子、表达载体、试剂盒 - Google Patents
一种抗达氟沙星单链抗体、核酸分子、表达载体、试剂盒 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,具体涉及一种抗达氟沙星单链抗体、核酸分子、表达载体、试剂盒。本发明提供的单链抗体,具有区别与同类单链抗体的氨基酸序列结构,对达氟沙星具有结合特异性和灵敏性,通过icELISA检测证实本发明提供的单链抗体的半数抑制浓度为19.47 ng/mL,与恩诺沙星,环丙沙星,沙拉沙星以及左氧氟沙星这4种结构相近的氟喹诺酮类药物无明显交叉反应。通过检测脱脂奶粉中的DAN标准品的添加回收率,测得DAN‑scFv的回收率在87.22%~103.44%之间。本发明进一步提供了突变的单链抗体,更进一步提高其特异性、灵敏性以及应用于达氟沙星检测的准确性。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种抗达氟沙星单链抗体、核酸分子、表达载体、试剂盒。
背景技术
DAN是治疗畜禽呼吸系统和泌尿系统感染等疾病的理想药物,目前被广泛用于兽医临床治疗。该药在吸收后主要以原型药物的形式在肝脏代谢,通过肾脏排出体外。然而,滥用该药物会造成其在动物组织及产品中大量残留,残留的DAN进入人体后,将会破坏肠道菌群的生态平衡,增加腹泻、结肠炎、过敏等疾病的发病概率;严重时还会引起呼吸系统、消化系统和泌尿系统的不可逆性损害;最为致命的是,无节制地使用抗生素将会逐渐增强致病菌的耐药性,使人类在面对细菌感染时进入无药可用的境地,与此同时还会破坏自然微生态平衡,造成严重的环境污染。
目前用于检测DAN残留的方法有:微生物法、免疫分析法、高效液相色谱法(LC)、液相色谱-质谱(LC-MS)法和酶联免疫吸附分析(ELISA)。目前,用于免疫检测的抗体都是单克隆抗体或多克隆抗体。单克隆抗体或多克隆抗体的制备必须通过细胞培养获得,整个生产过程复杂,消耗时间长,费用高,且不易进行操作。单链抗体是将抗体重链可变区和轻链可变区基因通过一个短肽链连接后融合表达出来的抗体片断,其表达形式主要有融合表达、胞内表达和分泌表达三种形式,和完整抗体相比,单链抗体具有以下优点:1)分子量小,大小仅为完整抗体的六分之一,免疫原性较低;2)组织穿透力强,容易进入实体瘤周围的微循环;3)血液清除快,肾脏蓄积很少;4)无Fc段,非特异结合低;5)易于通过基因工程大量生产;6)易于基因操作,更易构建重组免疫毒素。
因此,创新研发出抗达氟沙星单链抗体,能够进一步促进DAN残留的准确、高效、便捷检测。
发明内容
本发明的目的之一在于提供抗达氟沙星单链抗体,能够特异性结合达氟沙星,应用于达氟沙星药物残留检测。
本发明的目的之二在于提供抗达氟沙星单链抗体编码基因的核酸分子,用于制备本发明提供的单链抗体。同时,本发明还提供包含该核酸分子的重组载体、重组表达系统等用于制备本发明提供的单链抗体。
另外,本发明的目的还在于提供抗达氟沙星单链抗体在检测达氟沙星方面的应用于,具体可以用于制备检测试剂盒的原料,作为检测试剂或者作为载体包被原。
为了实现上述发明目的,本发明采用的技术方案如下:
一种抗达氟沙星单链抗体,由重链可变区、连接重链可变区和轻链可变区的短肽、轻链可变区依次连接组成,所述重链可变区的氨基酸序列如SEQ NO.1所示;轻链可变区的氨基酸序列如SEQ NO.2所示
为了进一步提高单链抗体结合达氟沙星的特异性和亲和力,所述重链可变区自N端起第47位氨基酸由Glu突变为Tyr;可选的,所述轻链可变区自N端第1位氨基酸由Asp突变为Ile;进一步可选的,所述轻链可变区自N端第103位氨基酸由Cys突变为Tyr。
可选的,所述连接重链可变区和轻链可变区的短肽为GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer。本发明提供的单链抗体的完整氨基酸序列如SEQ NO.3所示;更进一步为了提高单链抗体的性能,突变改性后的单链抗体的氨基酸序列如SEQ NO.4所示。
本发明通过基因重组表达的方式制备上述单链抗体,首先根据单链抗体的氨基酸序列设计其编码基因核苷酸序列,作为举例说明,编码上述未突变的单链抗体的核酸分子的核苷酸序列如SEQ NO.5所示。
具体的,在合成编码基因后,通过酶切连接技术构建重组载体,将重组载体转染DH5α等用于可用于重组蛋白表达的细胞系中构建重组表达系统,用于分泌表达制备上述单链抗体。
能够理解的是,上述单链抗体能够应用于检测达氟沙星,可以用于制备达氟沙星检测用免疫试剂盒,一种产品形式为:试剂盒中包括达氟沙星半抗原的酶标记物,上述单链抗体;其中上述单链抗体作为包被原;
第二种为:试剂盒中包括达氟沙星半抗原与载体蛋白的偶联物、上述单链抗体试剂和酶标记抗抗体;其中达氟沙星半抗原与载体蛋白的偶联物作为包被原;
第三种为:试剂盒中包括达氟沙星半抗原的酶标记物、上述单链抗体试剂和抗抗体;其中抗抗体作为包被原;
第四种为:试剂盒中包括达氟沙星半抗原与载体蛋白的偶联物、上述单链抗体的酶标记物;其中达氟沙星半抗原与载体蛋白的偶联物作为包被原;
第五种为:所述试剂盒包括胶体金试纸,包括样品吸收垫、胶体金垫、反应膜和吸水垫,其依次连接;胶体金垫包被有胶体金标记的上述单链抗体;所述反应膜上含有检测带和质控带,检测带位置包被有达氟沙星半抗原与载体蛋白的偶联物,质控带位置包被有鼠抗His标签单克隆抗体。
上述试剂盒还包括标准品溶液、洗涤液、缓冲液等。
本发明提供的抗达氟沙星单链抗体(DAN-scFv),具有区别与同类单链抗体的氨基酸序列结构,对达氟沙星具有结合特异性和灵敏性,本发明通过icELISA检测证实本发明提供的单链抗体的半数抑制浓度(half maximal inhibitory concentration,IC50)为19.47ng/mL,与恩诺沙星,环丙沙星,沙拉沙星以及左氧氟沙星这4种结构相近的氟喹诺酮类药物无明显交叉反应。通过检测脱脂奶粉中的DAN标准品的添加回收率,测得DAN-scFv的回收率在87.22%~103.44%之间。
为了更进一步提高其特异性、灵敏性以及应用于达氟沙星检测的准确性,本发明进一步提供了突变的单链抗体,对轻链可变区的特定氨基酸结构进行突变,具体为重链可变区自N端起第47位氨基酸由Glu突变为Tyr;轻链可变区自N端第1位氨基酸由Asp突变为Ile;自N端第104位氨基酸由Arg突变为Tyr。突变后的单链抗体与DAN结合能明显降低,降低至-11.3kcal/mol,。icELISA鉴定结果表明,突变体单链抗体的IC50为15.74ng/mL,比亲本抗体的灵敏度提高了1.3倍。
附图说明
图1是实施例1中琼脂糖凝胶电泳图VH、VL的扩增结果示意图;
图2是实施例1中原核表达筛选到的scFv阳性克隆基因的扩增片段电泳图;
图3是实施例2中DAN-scFv-01基因表达的蛋白SDS-PAGE检测图;
图4是实施例2中DAN-scFv-01基因表达的蛋白Western Bloting检测图;
图5是DAN-scFv-01蛋白建模结果;
图6是DAN-scFv-01与DAN的分子对接结果;
图7是实施例3中DAN-scFv-02基因表达的SDS-PAGE检测图。
具体实施方式
未特别指明的,实施例及试验例中相关操作均为领域内常规技术手段,比如参照Sambrook等编著的分子克隆实验手册(Sambrook J&Russell DW.Molecular cloning:alaboratory manual.2001),或者产品制造厂商提供的说明书等。
实施例及试验例中所用各类培养基、试剂、质粒、细胞系、工具酶等均为市售商品;
分泌抗达氟沙星单克隆抗体的杂交瘤细胞为实验室通过设计完全抗原,通过动物免疫的方式筛选获得并保存。
下述实施例仅对本发明作进一步详细说明,但不构成对本发明的任何限制。
实施例1抗达氟沙星单链抗体筛选与构建。
具体步骤为:
1)从液氮罐中快速取出实验室保存的可分泌抗达氟沙星单克隆抗体的杂交瘤细胞,在无菌环境下置于37℃恒温水浴锅中缓慢晃动约2min使其迅速解冻;将解冻的杂交瘤细胞以1000rpm的速度离心10min,保留沉淀;然后再加入5mL 10%DMEM完全培养基重悬细胞沉淀,最后将重悬的细胞置于37℃,5%CO2恒温培养箱中观察培养,并在细胞生长旺盛时对细胞进行传代以保证细胞活力;当细胞数量生长到1×10 6个/瓶,选取生长状态良好的细胞提取细胞总RNA;
2)在超净台中,按照FastPure Cell/Tissue Total RNA Isolation Kit V2试剂盒说明书提取杂交瘤细胞总RNA,并用微量UV-Vis分光光度计测量RNA的浓度。按照PrimeScriptTMRT Master Mix(Perfect Real Time)试剂盒进行反转录以合成第一链cDNA;
3)以cDNA为模板,利用PCR技术扩增抗体的VH基因和VL基因,使用1.2%琼脂糖凝胶电泳检测扩增结果,如图1所示。使用Gel Extraction Kit D2500胶回收试剂盒对VH基因和VL基因片段进行回收纯化,纯化的DNA置于-20℃保存备用;
4)以纯化后的VH和VL基因片段为模板,通过重叠延伸PCR技术,以GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer为连接短肽,将VH和VL拼接成scFv,然后用1.2%琼脂糖凝胶电泳检测分析PCR扩增结果,如图2所示。使用胶回收试剂盒回收纯化scFv。上述纯化的PCR反应产物中补加含有SfiⅠ酶切位点的VH上游引物和含有VL下游引物,然后继续进行PCR扩增反应;扩增结束后胶回收纯化scFv基因;
5)将上一步中获得的纯化DNA(scFv基因)与pMD19-T Vector载体在PCR管中配制后于16℃过夜连接。将连接结果转入DH5α细胞中,挑取20个单菌落进行扩大培养,然后以pMD19-T F与pMD19-T R为引物进行菌液PCR鉴定,并将阳性菌液送去上海生工生物工程有限公司进行测序鉴定,获得抗达氟沙星单链抗体(DAN-scFv)编码基因序列,如SEQ NO.5所示,其中重链可变区的氨基酸序列如SEQ NO.1所示;轻链可变区的氨基酸序列如SEQ NO.2所示;重链可变区和轻链可变区通过GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer短肽依次连接;构建的DAN-scFv的完整氨基酸序列如SEQ NO.3所示。
实例2
本实施例制备抗达氟沙星单链抗体,标记为DAN-scFv-01,具体步骤为:
1)按照SEQ NO.5所示核苷酸序列合成核酸分子,作为scFv编码基因;
2)设计引物在在步骤1)提供的scFv编码基因两端连接XhoⅠ与HindⅢ酶切位点,引物如表1所示:
表1
下划线斜体字为XhoⅠ与HindⅢ酶切位点。
3)对pET-22b质粒与步骤2)提供的scFv编码基因进行Xho I和Hind III双酶切处理。将双酶切后的scFv编码基因与pET22b载体进行连接,构建pET22b-scFv重组质粒;
4)将步骤3)构建的pET22b-scFv重组质粒转染至DE3(BL21)表达菌株,发酵表达。
表达结果表征:重组表达的单链抗体DAN-scFv-01蛋白含有His-tag,采用Ni NTASepharose 6FF的预装柱(采购自北京索莱宝股份有限公司)对蛋白进行纯化,纯化后再进行蛋白超滤,对收集的洗脱液进行SDS-PAGE以及Western Bloting分析,蛋白大小约为26kD,结果如图3和图4所示,其中图3中的1表示16℃下重组表达的包涵体表达情况,2表示16℃下重组表达的可溶性表达情况;3表示30℃下重组表达的包涵体表达情况,4表示30℃下重组表达的可溶性表达情况;图3结果显示重组蛋白表达的最适温度条件为16℃;图4为16℃温度条件下,不同发酵表达时间的蛋白表达情况,其中1表示BL21原始菌包涵体表达情况,2表示BL21原始菌可溶性表达情况;3表示pET22b空载状态下包涵体表达情况,4表示pET22b空载状态下可溶性表达情况;5表示重组表达8h下包涵体表达情况;6表示重组表达16h下包涵体表达情况;7表示重组表达24h包涵体表达情况;8表示重组表达8h下可溶性蛋白表达情况;9表示重组表达16h下可溶性蛋白表达情况;10表示重组表达24h下可溶性蛋白表达情况。图3和图4的结果表明重组蛋白表达的最适条件为16℃,16h。
实施例3
本实施例构建并制备突变的抗达氟沙星单链抗体,标记为DAN-scFv-02
其构建过程包括通过Swiss-Model同源模拟程序建立DAN-scFv的3D模拟结构,如图6所示,使用结构分析和验证服务器对建模结果进行评价,选取打分通过的受体模型模拟与DAN分子对接,如图7所示,筛选确定DAN-scFv与DAN之间的相互作用的氨基酸位点,设计突变方案,最终确定的突变方案为重链可变区自N端起第47位氨基酸由Glu突变为Tyr;轻链可变区自N端第1位氨基酸由Asp突变为ILE;自N端第104位氨基酸由Arg突变为Tyr,构建形成DAN-scFv-02,DAN-scFv-02的完整氨基酸序列如SEQ NO.4所示。
制备表达DAN-scFv-02:按照实施例2同样的方法,根据SEQ NO.4所示氨基酸序列,设计编码基因序列,如SEQ NO.6所示,合成编码基因序列,构建重组质粒,并构建重组表达系统,表达制备DAN-scFv-02,SDS-PAGE表征结果如图7所示。
试验例性能检测
1、半数抑制浓度:
试验方法:以DAN的偶联蛋白为底物包被酶标板,将DAN-scFv与梯度稀释DAN标准品溶液加入孔中反应,通过HRP标记的抗His标签的鼠源单克隆抗体进行显色。使用酶标仪检测OD450的吸光值,以DAN标准品浓度与吸光值为x,y值构建抑制率曲线,计算其半数抑制浓度。
试验结果:icELISA检测证实,DAN-scFv-01的IC50=19.47ng/mL;DAN-scFv-02的IC50=15.74ng/mL,相比亲本抗体DAN-scFv-01,突变优化后的抗体DAN-scFv-02的灵敏度提高了1.3倍。
2、DAN结合回收率:
试验方法:使DAN标准品构建标准曲线。配置5%的脱脂牛奶,将1mg/mL DAN标准品溶液梯度稀释到100ng/mL,50ng/mL,10ng/mL。通过间接竞争ELISA 和标准曲线计算得出加标回收物质的浓度。通过公式回收率(%)=(测得的加标回收物质浓度/加标物质浓度)×100%计算其回收率。
试验结果:通过检测脱脂奶粉中的DAN标准品的添加回收率,测得DAN-scFv-01的回收率在87.22%~103.44%之间;DAN-scFv-02的回收率在80.31%~110.92%之间。
3、DAN结合能:
试验方法:采用Auto Dock Vina 1.1.2软件进行分子对接工作,在对接开始之前,使用Pymol学术开源版对受体蛋白结构进行处理并进行加氢。进而使用ADFR 1.0将所有处理好后的小分子以及受体蛋白转换为Auto Dock Vina 1.1.2对接必须的PDBQT格式。对接前,以蛋白的质心为盒子中心,调整合适的X、Y、Z边长构建盒子并使其充分包裹整个蛋白。对接时,将网格盒子以及处理好后的蛋白和小分子的PDBQT文件作为输入文件,使用Vina进行对接,对接全局搜索的详尽度设为32,其余参数保持默认设置。最后,输出的打分最高的对接构象被我们认为是结合构象。其结合能视为该抗体与受体的结合能。
试验结果:DAN-scFv-01与DAN的结合能为-7.4kcal/mol;DAN-scFv-02与DAN的结合能-11.3kcal/mol,突变优化后的抗体与DAN之间的结合能降低。
4、特异性:
试验方法:通过icELISA测定DAN-scFv对多种氟喹诺酮类药物的抑制率,并根据公式CR(%)=IC50(其他氟喹诺酮类类药物)/IC50(DAN)×100%计算交叉反应率(CR)。
试验结果:
表1DAN-scFv-01与其他四种氟喹诺酮类药物的交叉反应
表2 DAN-scFv-02与其他氟喹诺酮类药物的交叉反应
与恩诺沙星,环丙沙星,沙拉沙星以及左氧氟沙星这4种结构相近的氟喹诺酮类药物无明显交叉反应
整体结论:本发明提供的抗达氟沙星单链抗体特异性结合DAN,能够灵敏的捕获DAN,能够应用于达氟沙星残留检测,用于制备达氟沙星检测的试剂盒,比如酶免疫试剂盒或胶体金试剂盒。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.一种抗达氟沙星单链抗体,由重链可变区、连接重链可变区和轻链可变区的短肽、轻链可变区依次连接组成,其特征在于,所述重链可变区的氨基酸序列如SEQ NO.1所示;轻链可变区的氨基酸序列如SEQ NO.2所示。
2.如权利要求1所述的抗达氟沙星单链抗体,其特征在于,所述重链可变区自N端起第47位氨基酸由Glu突变为Tyr;
可选的,所述轻链可变区自N端第1位氨基酸由Asp突变为Ile;
进一步可选的,所述轻链可变区自N端第104位氨基酸由Arg突变为Tyr。
3.如权利要求1~2任一项所述的抗达氟沙星单链抗体,其特征在于,所述连接重链可变区和轻链可变区的短肽为GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer。
4.一种核酸分子,其特征在于,如权利要1所述的单链抗体的编码基因。
5.如权利要求4所述核酸分子,其特征在于,其核苷酸序列如SEQ NO.5所示。
6.一种含有如权利要求4或5所述的核酸分子的重组载体。
7.一种含有如权利要求8所述的重组载体的重组表达系统。
8.一种如权利要求1~3任一项所述的单链抗体的应用,其特征在于,用于检测达氟沙星。
9.一种用于检测达氟沙星的试剂盒,其特征在于,包括含有如权利要求1~3任一项所述单链抗体的抗体试剂,或者以如权利要求1~3任一项所述单链抗体作为包被原的载体,或者含有如权利要求1~3任一项所述的单链抗体酶标记物的试剂;
可选的还包括达氟沙星半抗原与载体蛋白的偶联物、或达氟沙星半抗原的酶标记物,和酶标记抗抗体或抗抗体。
10.一种用于检测达氟沙星的试剂盒,其特征在于,包括样品吸收垫、胶体金垫、反应膜和吸水垫,其依次连接;所述胶体金垫包被有胶体金标记的权利要求1~3任一项所述单链抗体;所述反应膜上含有检测带和质控带,检测带位置包被有达氟沙星半抗原与载体蛋白的偶联物,质控带位置包被有鼠抗His标签单克隆抗体。
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