Screening and identification of single-chain fragment variable against the receptor-binding domain of spike protein of SARS-CoV-2 / 中国生物制品学杂志

@#Objective To construct a single-chain fragment variable(scFv)phage display library against receptor-binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(S)to screen specific scFv and identify the function.Methods m RNA was extracted from spleen cells of mice immunized with RBD protein and reversely transcribed into c DNA,with which as template,genes of the hight chain fragment of variable(VH)and light chain fragment of variable(VL)of scFv were amplified and then assembled into scFv gene fragment through splicing overlap extension PCR(SOE-PCR).The scFv gene fragment was inserted to phage vector to construct scFv phage display library.After four rounds of biopanning,the scFv gene with strong binding ability to RBD was screened and expressed recombinantly,purified and identified for biological activity.Results The constructed scFv phage library showed a titer of 6.0×10(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(334～353)),RBD9(S(334～353)),RBD9(S(439～458))and RBD13(S(439～458))and RBD13(S(499～518)).Homologous model of scFv constructed by online server SWISS-MODEL showed a good quality and was used for molecular docking.The interface at which scFv11 interacted with RBD only partially coincided with the interaction interface of human angiotensin converting enzyme 2(ACE2)and RBD,and the interaction interfaces of scFv12 and scFv25 with RBD were quite different from that of ACE2.Conclusion In this study,scFv specifically bound to SARS-Co V-2 RBD was screened and prepared through constructing scFv phage library against SARS-CoV-2 RBD,which provided experimental basis for further development of anti-SARS-CoV-2 drugs and detection reagents.

Desenvolvimento de teste diagnóstico não invasivo capaz de detectar anticorpos anti-SARS-CoV-2 em urina / Development of a non-invasive diagnostic test capable of detecting anti-SARS-CoV-2 antibodies in urine

A plataforma de ELISA (ensaio de imunoabsorção por ligação enzimática) tem sido amplamente utilizada para detectar anticorpos anti-SARS-CoV-2 gerados após a exposição ao vírus ou à vacinação. A amostra comumente utilizada para a realização do teste é o soro. Até o momento, nenhum estudo havia investigado a urina do paciente como amostra para detectar anticorpos específicos para o vírus SARS-CoV-2. A urina é um espécime biológico que traz vantagens significativas inerentes ao tipo de amostra, que compreende coleta não invasiva, de fácil manuseio e armazenamento. Neste trabalho, propomos um ELISA indireto in house baseado no uso de urina e proteínas recombinantes do Nucleocapsídeo (N) ou da Spike (S) do vírus SARS-CoV-2. As proteínas recombinantes (r) de SARS-CoV-2, N e as subunidades da proteína S (S-Glic, S1-NGlic e RBD-NGlic), foram avaliadas usando um painel composto por aproximadamente 200 amostras de urina e de soro. A presença de anticorpos anti-SARS-CoV-2 na urina foi detectada com sensibilidade e especificidade similares ou superiores ao soro, nas quais foram obtidos valores de sensibilidade de 94,0%, 75,0%, 81,38% e 89,66%, e especificidade de 100%, 96,0%, 96,77% e 96,77%, frente às proteínas rSARS-CoV-2 N, S-Glic, S1-NGlic e RBDNGlic, respectivamente. Dessa forma, os dados apresentados sugerem que a urina poderia ser considerada como uma potencial amostra biológica para aplicação em plataformas de imunodiagnóstico para a infecção por SARS-CoV-2, trazendo benefícios tanto no contexto individual quanto populacional.

The Enzyme-linked immunosorbent assay (ELISA) method has been widely used to detect anti-SARS-CoV-2 antibodies generated after exposure to the virus or vaccination. The sample usually used to perform the test is the serum. Thus far, no study has investigated the urine of patients as biological sample to detect specific SARS-CoV-2 antibodies. Urine is a biological specimen with significant advantages inherent to the type of sample, which comprises non-invasive collection, easy handling and storage. In this work, we propose an in house urine-based indirect ELISA using recombinant proteins from Nucleocapsid (N) and Spike (S) of the SARSCoV-2 virus. SARS-CoV-2 recombinant N and S protein subunits (Gly-S, NonGly-S1 and NonGly-RBD) were evaluated in an ELISA platform with a panel composed about 200 urine and serum samples. The presence of anti-SARS-CoV-2 antibodies in urine was detected with similar or superior sensitivity and specificity to serum, in which sensitivity values of 94.0%, 75.0%, 81.38% and 89.66% were obtained, while specificity values were of 100.0%, 96.0%, 96.77% and 96.77%, respectively, against rSARS-CoV-2 N, S-Glic, S1-NGlic and RBD-NGlic proteins. In conclusion, the data presented suggest that urine could be considered as a potential biological sample for application in immunodiagnostic platforms for SARS-CoV-2 infection, with benefits to the individual and population context.

The SARS-CoV-2 Antibody and Drug Research Based on the Structure of the Spike Protein and the Mechanism of Cell Entry / 中国生物化学与分子生物学报

COVID-19 is a severe acute respiratory syndrome caused by a novel coronavirus, SARS-CoV- 2.COVID-19 is now a pandemic, and is not yet fully under control.As the surface spike protein (S) mediates the recognition between the virus and cell membrane and the process of cell entry, it plays an important role in the course of disease transmission.The study on the S protein not only elucidates the structure and function of virus-related proteins and explains their cellular entry mechanism, but also provides valuable information for the prevention, diagnosis and treatment of COVII)-19.Concentrated on the S protein of SARS-CoV-2, this review covers four aspects: (1 ) The structure of the S protein and its binding with angiotensin converting enzyme II (ACE2) , the specific receptor of SARS-CoV-2, is introduced in detail.Compared with SARS-CoV, the receptor binding domain (RBD) of the SARS-CoV- 2 S protein has a higher affinity with ACE2, while the affinity of the entire S protein is on the contrary.(2) Currently, the cell entry mechanism of SARS-CoV-2 meditated by the S protein is proposed to include endosomal and non-endosomal pathways.With the recognition and binding between the S protein and ACE2 or after cell entry, transmembrane protease serine 2(TMPRSS2) , lysosomal cathepsin or the furin enzyme can cleave S protein at S1/S2 cleavage site, facilitating the fusion between the virus and target membrane.(3) For the progress in SARS-CoV-2 S protein antibodies, a collection of significant antibodies are introduced and compared in the fields of the target, source and type.(4) Mechanisms of therapeutic treatments for SARS-CoV-2 varied.Though the antibody and medicine treatments related to the SARS-CoV-2 S protein are of high specificity and great efficacy, the mechanism, safety, applicability and stability of some agents are still unclear and need further assessment.Therefore, to curb the pandemic, researchers in all fields need more cooperation in the development of SARS-CoV-2 antibodies and medicines to face the great challenge.