搜索
>公司动态 > 新冠专区 >藏在新冠 (SARS-CoV-2) 突变毒株亲和力升高里的秘密

藏在新冠 (SARS-CoV-2) 突变毒株亲和力升高里的秘密

2020-04-27 16:51

SARS-CoV-2的棘突蛋白受体结合域(RBD)是病毒嗜性和感染性的关键因素[1,2]。最新研究分析了全球SARS-CoV-2毒株的基因组,发现32种RBD突变毒株。依据RBD突变位点,以上突变毒株可以分为10种突变类型。其中在武汉,深圳,香港和法国流行的三种突变毒株的棘突蛋白受体结合域(RBD)显示出增强的结构稳定性和更高的受体亲和力,表明这些突变病毒可能已增强了对人类的感染力[3]




SARS-CoV-2突变毒株分布及RBD突变分型



Fig.1 SARS-CoV-2病毒种类及分布,红色为亲和力增强型毒株;

黄色突变毒株亲和力与原型毒株无明显差异


由于RBD是结合人ACE2并启动细胞进入的唯一结构域,通常认为RBD应高度保守。但是多态性和差异性分析表明,与S蛋白的其他区域类似,RBD序列也呈现多样性[3]


已公布基因组序列的1609个SARS-CoV-2毒株中,经多态性分析发现有32个毒株在RBD中含有氨基酸突变。这些毒株来自多个地区,包括中国,英国,芬兰,法国,比利时,美国和印度。大多数突变体与最先发现的基因组(SARS-CoV-2 WuHan-Hu-1)相比,仅相差一个氨基酸。进一步分析得出32个RBD突变型分为10种类型(Fig. 1)[3]



三种RBD突变类型与人ACE2受体结合力提高



利用分子动力学模拟分析比较原型毒株SARS-CoV-2(SARS-CoV-2 Wuhan-Hu-1)RBD蛋白和突变型毒株的RBD蛋白与人ACE2的结合能力。分析结果显示与原型毒株RBD蛋白相比,三个RBD突变蛋白(N354D&D364Y,V367F,W436R)的ΔG均为-200 kJ / mol,比原型毒株低约25%,与人ACE-2的亲和力显著提高[3]



RBD突变的结构解析



RBD与ACE2的结合界面主要是无规卷曲构象,因而缺乏结构上的刚性。通过增加骨架的刚性可以维持相互作用表面的结合构象,从而提高亲和力。以残基510-524为中心的β折叠结构就起到了这种支架的作用,该区域附近的突变体N354D,D364Y,V367F和W436R在结构上增加了刚性作用,同时这些突变位点导致结合位点区域的ΔG总体降低,增加了结构的稳定性,这些因素共同导致了突变体RBD与ACE2亲和力的提高。


另外,ACE2表面附近带大量负电荷,而突变体W436R提供了带正电的氨基酸。这种潜在的静电作用力可能有助于亲和力的提高。

Fig. 2 RBD突变体的结构分析及其结合亲和力的影响:

突变氨基酸和片段510-524的空间位置



针对文章中提到RBD突变体与ACE2蛋白的亲和力增强这一观点,ACROBiosystems用突变型和原型RBD与ACE2的结合活性做了ELISA、SPR、BLI三种技术的验证。




ELISA验证



ACROBiosystems SARS-CoV-2 RBD突变蛋白验证ELISA Binding Assay结果显示,所有RBD突变蛋白与人ACE2受体蛋白结合活性均高于原型毒株RBD蛋白, RBD (V367F) EC50: 0.31nM > RBD (W436R) EC50: 0.89nM > RBD (N354D, D364Y) EC50: 0.97nM > 原型毒株RBD EC50: 1.47nM (Fig. 3)。

Fig. 3 Human ACE2与 SARS-CoV-2 RBD蛋白结合活性ELISA比较





BLI验证


BLI验证发现,突变蛋白 RBD(V367F),RBD (W436R)和RBD (N354D, D364Y) 与ACE2的亲和力均达到nM水平, KD值分别为5.5nM, 6.85nM和6.32nM,比原型毒株RBD与ACE2 受体蛋白亲和力(24.4nM)高3倍以上(Fig. 4)。


Fig. 4 BLI技术验证Human ACE2与 SARS-CoV-2 RBD蛋白亲和力



Bioactivity- SPR



与BLI检测结果类似,SPR原型毒株RBD与人ACE2受体蛋白亲和力为13.1nM,突变蛋白RBD(V367F),RBD (W436R) 和RBD (N354D, D364Y)的KD值分别为4.33nM、6.96nM和7.57nM(Fig. 5)。


Fig. 5 SPR技术验证Human ACE2与 SARS-CoV-2 RBD蛋白亲和力



根据ELISA、SPR、BLI技术的结合活性验证发现,突变型RBD(V367F)、RBD (W436R) 和RBD (N354D, D364Y)与ACE2的亲和力均高于原型毒株,与文章中结构分析结果一致。


>>>>新冠相关产品列表

Kits  For research use only.
Molecule Cat. No. Species Product Description Protocol/Order
S protein RBD EP-105 SARS-CoV-2 SARS-CoV-2 Inhibitor Screening Kit

订购

S1 protein MBS-K001 SARS-CoV-2 SARS-CoV-2 Spike S1 protein-coupled magnetic beads

订购

S protein RBD MBS-K002 SARS-CoV-2 SARS-CoV-2 Spike protein RBD-coupled magnetic beads

订购

TAS-K003 SARS-CoV-2 Anti-SARS-CoV-2 neutralizing antibody titer serologic assay kit

订购

S1 protein SP-12 SARS-CoV-2 SARS-CoV2 Spike S1 protein-Coated Plates, Clear, 96-Well

订购

S protein RBD RP-13 SARS-CoV-2 SARS-CoV2 Spike protein RBD-Coated Plates, Clear, 96-Well

订购

S1 protein TAS-K001 SARS-CoV-2 SARS-CoV-2 antibody titer assay kit (Spike protein S1)

点击预购

S protein RBD TAS-K002 SARS-CoV-2 SARS-CoV-2 antibody titer assay kit (Spike protein RBD)

点击预购

Antibody
Molecule Cat. No. Species Product Description Protocol/Order
S protein RBD SAD-S35 Human Anti-SARS-CoV-2 neutralizing antibody (Human IgG1)

订购

Proteins
Molecule Cat. No. Species Host Product Description Protocol/Order
ACE2 AC2-H82E6 Human HEK293 Biotinylated Human ACE2 / ACEH Protein, His,Avitag™ (MALS verified)

订购

AC2-H82F9 Human HEK293 Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag™

订购

AC2-H5257 Human HEK293 Human ACE2 / ACEH Protein, Fc Tag (MALS verified)

订购

AC2-C52H7 Cynomolgus HEK293 Cynomolgus ACE2 / ACEH Protein, His Tag

订购

AC2-H52H8 Human HEK293 Human ACE2 / ACEH Protein, His Tag (MALS verified)

订购

AC2-R5246 Rat HEK293 Rat ACE2 / ACEH Protein, His Tag (MALS verified)

订购

AC2-M5248 Mouse HEK293 Mouse ACE2 / ACEH Protein, His Tag (MALS verified)

订购

AC2-P5248 Paguma larvata HEK293 Paguma larvata ACE2 / ACEH Protein, His Tag

订购

S protein SPN-C52H4 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein (R683A, R685A), His Tag

订购

SPN-C52H8 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein (R683A, R685A) , His Tag, active trimer (MALS verified)

订购

SPN-S52H5 SARS HEK293 SARS S protein (R667A), His Tag

订购

S1 protein S1N-C52H3 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein, His Tag

订购

S1N-C82E8 SARS-CoV-2 HEK293 Biotinylated SARS-CoV-2 (COVID-19) S1 protein, His,Avitag™ (MALS verified)

订购

S1N-S52H5 SARS HEK293 SARS S1 protein, His Tag (MALS verified)

订购

S1N-C52H4 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein, His Tag (MALS verified)

订购

S1N-C5255 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein, Fc Tag

订购

S1N-C5257 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein, Mouse IgG2a Fc Tag

订购

S protein RBD SPD-C82E9 SARS-CoV-2 HEK293 Biotinylated SARS-CoV-2 (COVID-19) S protein RBD, His,Avitag™ (MALS verified)

订购

SPD-C5255 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD, Fc Tag (MALS verified)

订购

SPD-S52H6 SARS HEK293 SARS S protein RBD, His Tag (MALS verified)

订购

SPD-C52H3 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD, His Tag (MALS verified)

订购

SPD-C5259 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD, Mouse IgG2a Fc Tag

订购

SPD-S52H4 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (V367F), His Tag

订购

SPD-S52H5 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (N354D), His Tag

订购

SPD-S52H7 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (W436R), His Tag

订购

SPD-S52H8 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (R408I), His Tag

订购

SPD-S52H3 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (N354D, D364Y), His Tag

订购

Nucleocapsid protein NUN-C51H9 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) Nucleocapsid protein, His Tag

订购

NUN-C5227 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) Nucleocapsid protein, His Tag

订购

NUN-C81Q6 SARS-CoV-2 E.coli Biotinylated SARS-CoV-2 (COVID-19) Nucleocapsid protein, His,Avitag™

订购

Envelope protein ENN-C5128 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) Envelope protein, His Tag

订购

Papain-like Protease PAE-C5148 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) Papain-like Protease Protein, His Tag

订购

NSP1 NS1-C51H7 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP1 Protein, His Tag

订购

NSP7 NS7-C51H6 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP7 Protein, His Tag

订购

NSP8 NS8-C5149 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP8 Protein, His Tag

订购

NSP7 & NSP8 NS8-C5125 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP7&NSP8 Protein, His Tag

订购

S1 protein S1N-C5256 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein (D614G), His Tag

点击预购

预计上线时间:6月5日

SIN-V52H3 HCoV-NL63 HEK293 HCoV-NL63 S1 protein, His Tag

点击预购

SIN-V52H4 HCoV-229E HEK293 HCoV-229E S1 protein, His Tag

点击预购

SIN-V52H5 HCoV-OC43 HEK293 HCoV-OC43 S1 protein, His Tag

点击预购

SIN-V52H6 HCoV-HKU1(isolate N5) HEK293 HCoV-HKU1(isolate N5) S1 protein, His Tag

点击预购

S1 protein CTD S1D-C52H3 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein CTD, His Tag

点击预购

S1 protein NTD S1D-C52H6 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S1 protein NTD, His Tag

点击预购

S protein RBD SPD-C52H4 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (G476S), His Tag

点击预购

SPD-C52H5 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S protein RBD (V483A), His Tag

点击预购

S2 protein S2N-C5253 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S2 protein, Fc Tag

点击预购

预计上线时间:6月4日

S2N-C5254 SARS-CoV-2 HEK293 SARS-CoV-2 (COVID-19) S2 protein, Mouse IgG2a Fc Tag

点击预购

预计上线时间:6月2日

Envelope protein ENN-C51P3 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) Envelope protein, His,BBP Tag

点击预购

预计上线时间:6月12日

NSP15 NS5-C5145 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP15 Protein, His Tag

点击预购

预计上线时间:6月1日

NSP16 & NSP10 NS0-C51W3 SARS-CoV-2 E.coli SARS-CoV-2 (COVID-19) NSP16&NSP10 Heterodimer Protein, His Tag&Twin Strep Tag

点击预购

预计上线时间:6月1日




>>>>

更多新冠病毒蛋白和检测试剂盒信息请点击查看



您可通过以下方式联系到ACROBiosystems:

邮件:order.cn@acrobiosystems.com

电话:13521050293

微信:扫描下方二维码即可进行沟通

(请备注公司+姓名)


Reference:

1. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. March 2020:S0092-8674(20)30229–4. doi:10.1016/j.cell.2020.02.052

2. Chen Y, et al. Structure analysis of the receptor binding of 2019-nCoV. Biochem Biophys Res Commun. 2020;2(xxxx):0–5. doi:10.1016/j.bbrc.2020.02.071

3. Ou J, et al. Emergence of RBD mutations from circulating SARS-CoV-2 strains with enhanced structural stability and higher human ACE2 receptor affinity of the spike protein 2020; doi:https://doi.org/10.1101/2020.03.15.991844




消息提示

请输入您的联系方式,再点击提交!

确定