CN114478696A - 一种新型冠状病毒刺突蛋白受体结合区rbd的亲和多肽及其应用 - Google Patents
一种新型冠状病毒刺突蛋白受体结合区rbd的亲和多肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种新型冠状病毒刺突蛋白受体结合区RBD的亲和多肽及其应用。亲和多肽的氨基酸序列分别为ID:SHWYWWR,HWHFWPK,HWPYFHH,HSWWHRW,WHTHRPA,WHKWPRF;能够特异结合新型冠状病毒刺突蛋白受体结合区RBD。本发明多肽及包含该多肽的生物活性片段可以与其它能够治疗SARS‑CoV‑2病毒的药物形成抗病毒的药物复合物,也可以与检测探针材料形成用于病毒检测的复合探针,多肽与新冠病毒RBD结合力强,特异性高,为抗病毒药物与病毒检测试剂的研发提供了新选择。
Description
技术领域
本发明属于生物医学领域的一种多肽及其应用,具体是涉及了一种新型冠状病毒刺突蛋白受体结合区RBD的亲和多肽及其应用。
背景技术
一种新型冠状病毒SARS-CoV-2与另外两种高致病性病毒SARS-CoV、 MERS-CoV同属冠状病毒科β冠状病毒属,SARS-CoV-2病毒的基因组与 SARS-CoV具有80%的同源性。SARS-CoV-2病毒与SARS-CoV病毒相似,都使用外壳表面的刺突包膜糖蛋白(S蛋白)侵染并进入到宿主细胞中。病毒进入细胞由S蛋白的受体结合域(RBD)与其受体血管紧张素酶2(ACE2)结合开始,二者的作用导致S蛋白的S2亚基的构象发生变化,形成六个螺旋束,进而导致病毒和宿主细胞之间的膜融合。最新的研究显示,SARS-CoV-2病毒的RBD 包含免疫显性表位,可引发中和抗体,为SARS-CoV-2感染提供保护,免疫血清中存在的90%中和抗体的靶点都包含RBD。
因此,RBD部分蛋白质片段是开发SARS-CoV-2病毒特效药物和进行疫苗设计的优良靶点。
发明内容
针对目前新冠病毒的研究现状,本发明利用丝状噬菌体随机展示文库对新冠病毒S蛋白RBD进行亲和优化处理,获得了与RBD高亲和性的多肽序列,具有安全、实用、方便和高效的优势。同时,本发明将提供该亲和多肽在抗新冠病毒药物研发与病毒检测中的应用。
本发明采用以下技术方案。
一、一种亲和多肽:
所述亲和多肽的氨基酸序列选自SHWYWWR,HWHFWPK,HWPYFHH, HSWWHRW,WHTHRPA,WHKWPRF其中之一,具体可以如SEQ ID No.1~SEQ ID No.6。
所述亲和多肽能够特异结合新型冠状病毒(SARS-CoV-2)刺突蛋白受体结合区RBD。
所述亲和多肽的氨基酸序列分别为:SHWYWWR,HWHFWPK,HWPYFHH,HSWWHRW,WHTHRPA,WHKWPRF。如下表。
表1 RBD亲和多肽氨基酸序列表
二、一种生物活性片段:
所述的生物活性片段包含权利要求1所述亲和多肽,为生物活性片段中功能区的一部分;
所述的生物活性片段也具有和所述亲和多肽一致的功能,即能够与新型冠状病毒SARS-CoV-2病毒RBD结合。
生物活性片段在制备药物中应用。
三、一种核苷酸序列:所述核苷酸序列能编码所述亲和多肽。
四、一种核苷酸序列:所述核苷酸序列能编码所述生物活性片段。
五、一种抗病毒的药物复合物:所述药物复合物包含所述亲和多肽或者所述生物活性片段,也包含用于治疗新型冠状病毒SARS-CoV-2的药物或药物载体。
所述的多肽或生物活性片段作为靶向抑制剂,与能够治疗新型冠状病毒 SARS-CoV-2的药物化学连接或混合作为抗病毒的药物复合物,也可以与包裹这些抗病毒药物的载体化学连接或混合作为能够抗病毒的药物复合物。
所述的药物为穿入和脱壳抑制剂、DNA多聚酶抑制剂、蛋白质抑制剂、神经氨酸酶抑制剂、核苷类逆转录酶抑制剂、核苷类逆转录酶抑制剂或广谱抗病毒药物中的任意一种;
所述的药物载体为脂质体、天然高分子材料、人工合成的高分子材料、无机晶体材料、碳材料、聚合物囊泡、聚合物胶束中的任意一种。
六、一种用于病毒检测的复合探针:
所述复合探针同时包含所述亲和多肽或者所述生物活性片段和能用于病毒检测的探针材料。所述的多肽或生物活性片段作为病毒识别原件,与能够产生信号的探针材料化学连接或混合作为病毒检测的复合探针。
所述的探针材料为有机荧光小分子、有机高分子荧光材料、荧光蛋白,无机荧光材料、分子影像造影剂材料中的任意一种。
本发明在制备与生产用于检测、预防或治疗冠状病毒引起疾病的试剂或药物中应用。
所述冠状病毒为新型冠状病毒SARS-CoV-2及其变异株。
本发明的多肽特异性强,亲和能力高,且操作相对简便,适用于用来寻找新冠病毒S蛋白RBD的结合多肽。
本发明的多肽及包含该多肽的生物活性片段可以与其它能够治疗 SARS-CoV-2病毒的药物形成抗病毒的药物复合物,也可以与检测探针材料形成用于病毒检测的复合探针。本发明所述多肽与新冠病毒RBD结合力强,特异性高,为抗病毒药物与病毒检测试剂的研发提供了新选择。
与现有技术相比,本发明具有以下突出优势:
(1)本发明筛选出的多肽能特异结合SARS-CoV-2病毒S蛋白RBD,而不结合其他蛋白,具有专一性。
(2)本发明使用噬菌体展示技术筛选与SARS-CoV-2病毒S蛋白RBD结合的小分子多肽,具有耗时较短,操作简便,成功率高的特点,为研制治疗药物与检测试剂奠定基础。
(3)本发明筛选出的能与SARS-CoV-2病毒S蛋白RBD结合的小分子多肽,可以利用标准化的化学合成工艺进行合成,相比抗体类试剂,其生产、纯化、保存成本大大降低,而且不需要借助于动物,这使得它们与抗体开发技术相比更经济的同时没有伦理上的顾虑;
(4)本发明提供的多肽与SARS-CoV-2病毒S蛋白RBD的特异结合作用,为进一步寻找新的病毒治疗靶点,研究病毒RBD与细胞表面受体的相互作用,筛选更有价值的抗病毒药物与病毒检测试剂提供了参考。
附图说明
附表1为实施例1每轮噬菌体文库筛选后获得噬菌体滴度统计结果。
附表2为实施例1第3和第4轮噬菌体文库筛选获得亲和多肽频次统计结果。
附图1为实施例2噬菌体酶联免疫吸附实验(ELISA)结果统计图。
附图2为实施例3多肽竞争抑制实验结果统计图。
具体实施方式
下面通过实施例对本发明作进一步的详细说明,以下实施例是对本发明的解释而本发明并不局限于以下实施例。凡在本发明的精神和原则之内,所做的任何更改和变化,均应当包括在本发明的保护范围之内。
本发明的实施例如下:
实施例1:
利用噬菌体展示技术筛选特异结合SARS-CoV-2病毒S蛋白RBD的亲和多肽。
1)SARS-CoV-2病毒S蛋白RBD包被
a.利用NaHCO3(0.1M,pH8.6)溶液对病毒RBD重组蛋白进行稀释,终浓度为20μg/mL;
b.将RBD稀释液加到聚苯乙烯多孔板中,放入湿盒,4℃包被过夜。
2)噬菌体文库亲和筛选
a.封闭:包被了RBD的多孔板中加入NaHCO3+BSA(0.5mg/ml)的封闭液,室温震荡封闭1小时后,去掉封闭液,用TBST(TBS+0.5%(v/v)tween-20) 溶液洗涤3次;
b.噬菌体文库结合:Ph.D.TM-7噬菌体随机展示文库取2×1011pfu噬菌体用 TBST稀释后加入到多孔板中,室温震荡孵育1小时;
c.洗涤:用TBST洗涤液洗涤多孔板10次;
d.洗脱:除去孔板中的洗涤液并拍干,加入甘氨酸洗脱液(0.2M的甘氨酸 -盐酸,pH2.2,1mg/mL BSA),冰上孵育10分钟,将与RBD特异性结合的噬菌体洗脱下来。
e.中和:加入1M的Tris-HCl(pH9.1)中和液,混匀后pH为中性;
f.子库扩增:将中和后的噬菌体溶液加入到提前准备好的对数期ER2738 菌中,37℃剧烈震荡培养4.5小时。
g.噬菌体纯化:取扩增后的混合液离心,取上清加入六分之一体积的 PEG/NaCl试剂(20%PEG,3.5M NaCl)混合均匀,4℃过夜沉降后离心去上清,将沉淀用TBS重悬得到纯化后的噬菌体。
h:上述a~g步骤为第一轮筛选,重复这些步骤4次得到特异结合 SARS-CoV-2病毒S蛋白RBD的亲和噬菌体。
3)铺平板验证亲和噬菌体的富集程度:将每轮筛选得到的噬菌体溶液用 TBS进行梯度稀释,然后加入到过夜培养的大肠杆菌中,室温孵育5分钟,加入顶层琼脂后铺到含有IPTG/Xgal的固体LB板上,轻轻摇晃均匀。待液体完全凝固后,倒置放入37℃培养箱过夜培养。次日对平板上出现的噬菌斑进行计数,根据稀释倍数得到每轮筛选所得到的噬菌体数目。计数结果如附表1所示。
附表1每轮噬菌体文库筛选后获得噬菌体滴度统计
附表1的结果显示,在每轮筛选保持投入噬菌体数目相同(2×1011pfu)的情况下,输出噬菌体数目逐轮递增,第四轮达到了最大(1.20×108pfu),这表明亲和噬菌体在四轮的筛选过程中得到了有效地富集。
4)基因测序获得SARS-CoV-2病毒S蛋白RBD亲和多肽序列:对第3轮和第4轮得到的亲和噬菌体,铺平板后挑取单克隆扩增培养,提取质粒进行基因测序,从而获得噬菌体表面展示的亲和多肽序列。每轮多肽序列与频次统计如附表2所示。
附表2第3和第4轮噬菌体文库筛选获得亲和多肽频次统计
上表中可见,有6种亲和多肽在第3轮和第4轮筛选后合计重复次数大于1,其中SHWYWWR和HWHFWPK多肽重复次数相对更高,达到15次和30次。
测试1
噬菌体酶联免疫吸附实验(ELISA)验证并比较特异结合SARS-CoV-2病毒 S蛋白RBD的亲和多肽的结合力大小。
1)将亲和筛选后出现频率较高的噬菌体进行扩增并纯化。
2)利用NaHCO3(0.1M,pH8.6)包被液对病毒RBD重组蛋白进行稀释,终浓度为1μg/mL;将RBD稀释液加到96孔板中,放入湿盒,4℃包被过夜。
3)准备好的RBD用封闭液(PBS+0.5mg/ml BSA)封闭1小时。
4)去掉封闭液,加入用封闭液稀释好的具有不同插入片段的亲和噬菌体(2 ×109pfu)后室温孵育1小时。
5)每个孔用PBST(PBS+0.5%(v/v)Tween20)溶液洗涤5次,每次5分钟。
6)加入用PBS溶液稀释好的抗M13噬菌体抗体,37℃孵育1小时。每个孔用PBST洗涤3次,每次5分钟。
7)加入辣根过氧化物酶(HRP)标记的相应二抗,37℃孵育30分钟。每个孔用PBST洗涤3次,每次5分钟。
8)加入TMB显色液,室温孵育至充分显色后,加入2M硫酸终止液,酶标仪读取450nm波长处的吸光度值,统计所得结果见附图1。
测试2
多肽竞争抑制实验验证SARS-CoV-2病毒S蛋白RBD的亲和多肽结合的特异性。
1)将亲和筛选后出现频率最高的噬菌体进行扩增并纯化。
2)利用NaHCO3(0.1M,pH8.6)包被液对病毒RBD重组蛋白进行稀释,终浓度为1μg/mL;将RBD稀释液加到96孔板中,放入湿盒,4℃包被过夜。
3)准备好的RBD用封闭液(PBS+0.5mg/ml BSA)封闭1小时。
4)去掉封闭液,加入用封闭液稀释好的亲和噬菌体(2×109pfu)后室温孵育1小时。
5)每个孔用PBST(PBS+0.5%(v/v)Tween20)溶液洗涤5次,每次5分钟。
6)用PBS稀释化学合成好的多肽,并进行梯度稀释,序列与所用亲和噬菌体表面展示的外源多肽一致,室温孵育1小时。
7)每个孔用PBST(PBS+0.5%(v/v)Tween20)溶液洗涤5次,每次5分钟。
8)加入甘氨酸洗脱液(0.2M的Glycine-HCl,pH2.2,1mg/mL BSA),冰上孵育10分钟,收集洗脱下来的噬菌体,加入1M的Tris-HCl(pH9.1)中和液,混匀后pH为中性。
9)铺平板测定中和后的噬菌体洗脱液滴度,统计所得结果见附图2。
本发明涉及的基因和蛋白序列如下:
SEQ ID No.1;
名称:亲和多肽1的氨基酸序列
来源:人工序列(Artificial Sequence)
TCTCATTGGTATTGGTGGCGG
SEQ ID No.2;
名称:亲和多肽2的氨基酸序列
来源:人工序列(Artificial Sequence)
CATTGGCATTTTTGGCCTAAG
SEQ ID No.3;
名称:亲和多肽3的氨基酸序列
来源:人工序列(Artificial Sequence)
TGGCATACGCATAGGCCTGCTSEQ ID No.4;
名称:亲和多肽4的氨基酸序列
来源:人工序列(Artificial Sequence)
CATTGGCCTTATTTTCATCATSEQ ID No.5;
名称:亲和多肽5的氨基酸序列
来源:人工序列(Artificial Sequence)
TGGCATAAGTGGCCCAGGTTTSEQ ID No.6;
名称:亲和多肽6的氨基酸序列
来源:人工序列(Artificial Sequence)
CATTCGTGGTGGCATCGGTGG。
序列表
<110> 浙江大学
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Claims (9)
1.一种亲和多肽,其特征是:
所述亲和多肽的氨基酸序列选自SHWYWWR,HWHFWPK,HWPYFHH,HSWWHRW,WHTHRPA,WHKWPRF其中之一。
2.一种生物活性片段,其特征是:
所述的生物活性片段包含权利要求1所述亲和多肽。
3.权利要求1所述亲和多肽或者权利要求2所述生物活性片段的应用,其特征在于:在制备药物中的应用。
4.一种核苷酸序列,其特征在于:所述核苷酸序列能编码权利要求1所述亲和多肽。
5.一种核苷酸序列,其特征在于:所述核苷酸序列能编码权利要求2所述生物活性片段。
6.一种抗病毒的药物复合物,其特征在于,所述药物复合物包含权利要求1所述亲和多肽或者权利要求2所述生物活性片段,也包含用于治疗新型冠状病毒SARS-CoV-2的药物或药物载体。
7.一种用于病毒检测的复合探针,其特征在于:
所述复合探针同时包含权利要求1所述亲和多肽或者权利要求2所述生物活性片段和能用于病毒检测的探针材料。
8.权利要求1所述亲和多肽、权利要求2所述生物活性片段、权利要求4所述核苷酸序列、权利要求5所述核苷酸序列、权利要求6所述药物复合物、权利要求7所述复合探针的应用,其特征在于:在制备与生产用于检测、预防或治疗冠状病毒引起疾病的试剂或药物中的应用。
9.根据权利要求8所述的应用,其特征在于:
所述冠状病毒为新型冠状病毒SARS-CoV-2及其变异株。
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CN112321686A (zh) * | 2020-11-16 | 2021-02-05 | 北京大学深圳研究生院 | 一种靶向新冠肺炎病毒刺突蛋白的稳定多肽及其用途 |
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