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.

Objective To study the diagnostic value of MSCT in cystic lung cancer with ground glass opacity(GGO),and improve understanding of the disease as well as diagnostic accuracy.Methods 2 1 cases of pathologically confirmed cystic lung cancer with GGO were analyzed retrospectively,all patients were examined by MSCT.CT features of the GGO and the cysts,including bronchovascular convergence sign,speculation sign,pleural indentation sign,lobulation,thickness and uniformity of the cyst wall,septa and the location of the lesion were analyzed.Results The image findings of the 21 cases of the cystic lung cancer with GGO included:bronchovasular convergence sign in 6 cases(28.6%),speculation sign in 10 cases(47.7%),pleural indentation sign in 12 cases(57.1%),lobulation in 8 cases(38.1%),10 cases(47.7%)showed pure GGO.The cyst wall was(1.7±0.9)mm in thickness and the cyst was (13.2±7.0)mm in diameter.Cyst wall was uniform in 9 cases(42.9%),septa was seen in 14 cases(66.7%),cysts of 16 cases(76.1%)were located in the upper lobes of the lung,1 case(4.7%)had distinctly mediastinal lymph node metastasis.Air-fluid lever,mural nodule or intrapulmonary metastasis were not present in any of the cases.Conclusion The majority of the cystic lung cancer with GGO are adenocarcinoma.In addition of the interference of its prominent cystic features on MSCT,the cystic lung cancer have characteristics of lung cancer with GGO.By analyzing its CT features,diagnostic accuracy can be improved before operation.

Objective:To explore the genetic etiology of a fetus with Coffin-Siris syndrome2 (CSS2).Methods:A fetus with abnormal ultrasound findings detected at Luoyang Maternal and Child Health Care Hospital in July 2023 was selected as the study subject. Clinical data were analyzed retrospectively. Whole exome sequencing was carried out on fetal tissue and parental peripheral blood samples, and candidate variant was verified by Sanger sequencing and pathogenicity analysis. This study was approved by Medical Ethics Committee of the Luoyang Maternal and Child Health Hospital (Ethics No. LYFY-YCCZ-2023011).Results:Color Doppler ultrasound at 16 + gestational weeks revealed bilateral ventriculomegaly and cerebellar hypoplasia in the fetus. Trio-WES found that the fetus has harbored a heterozygous c. 553C>T (p.Gln185Ter) variant of the ARID1A gene, which was verified by Sanger sequencing to have a de novo origin. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c. 553C>T (p.Gln185Ter) variant of the ARID1A gene was classified as pathogenic (PVS1+ PS2+ PM2_Supporting). Conclusion:The fetus was diagnosed with CSS type 2, and the heterozygous c. 553C>T (p.Gln185Ter) variant of the ARID1A gene probably underlay its brain malformations.