Construction and identification of recombinant adenovirus expressing S protein receptor binding domain and N protein of SARS-CoV-2 Delta variant / 中国生物制品学杂志

@#ObjectiveTo construct and identify a recombinant adenovirus expressing S protein receptor binding domain(RBD)and N protein of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)Delta variant.MethodsThe RBD and N gene fragments of SARS-CoV-2 were cloned into pcDNA3.0BA vector respectively to construct recombinant plasmid pcDNA3.0BA-RBD-N. The RBD-CMV-N fragment was amplified by PCR and inserted into shuttle vector pShuttle-CMV. The shuttle plasmid pShuttle-RBD-N was then homologously recombined with pAdeasy-1 to obtain recombinant plasmid pAdeasy-1-RBD-N,which was transfected into HEK293 cells for recombinant adenovirus Ad-RBD-N packaging. The transcription of RBD and N genes of recombinant adenovirus in HEK293 cells was detected by RT-PCR,while the expre-ssion of RBD and N proteins by Western blot and immunofluorescence assay. 12 female BALB/c mice were immunized with Ad-RBD-N by intramuscular injection at a dose of 5 × 109copies per mouse. Blood samples were collected 14 d after immunization,and the serum antibody titers were measured by ELISA.ResultsThe RBD and N genes of recombinant adenovirus were transcribed normally in HEK293 cells,and the RBD and N proteins were expressed normally in MA104 cells. Mice immunized with the recombinant adenovirus produced specific IgG antibodies against RBD and N proteins.ConclusionThe recombinant adenovirus expressing S protein RBD and N protein of SARS-CoV-2 Delta variant was succe-ssfully constructed,which laid a foundation of the follow-up research on Delta variant vaccines.

Prokaryotic expression, purification and identification of the N protein of middle east respiratory syndrome coronavirus / 中华微生物学和免疫学杂志

Objective To express the N protein of middle east respiratory syndrome coronavirus ( MERS-CoV) in prokaryotic expression system and evaluate the immunogenicity of the purified recombinant N protein.Methods The gene encoding N protein of MERS-CoV was synthesized and cloned into the vector pET30a to construct the recombinant expression plasmid pET30a-MERS-CoV-N.The transformed E.coli BL21 ( DE3) strains carrying expression plasmid were induced by IPTG to express N protein.The expressed protein was purified by using affinity chromatography.SDS-PAGE and Western blot assays were used to iden-tify the expressed N protein and evaluate its immunogenicity.Results The recombinant N protein was suc-cessfully expressed in soluble form with the size of 46×103 .The results of Western blot assay showed that the recombinant N protein could specifically react with rabbit serum samples positive for antibodies against N protein B-cell epitope peptide and mouse anti-His tag antibodies.No positive band against N protein was found when primary antibody was used with thirty adult serum samples from Beijing in 2008.Conclusion N protein of MERS-CoV was successfully expressed in prokaryotic expression system.The successful expres-sion of N protein laid the foundation for immunological diagnosis of N protein of MERS-CoV and researches on its immunogenicity.

Caracterização genética dos vírus do sarampo genótipo D4 detectados no Brasil no período de 2003-2012 / Genetic characterisation of measles virus genotype D4 detected in Brazil, 2003-2012

O sarampo é uma doença exantemática viral, altamente infecciosa, causada por um vírus da família Paramyxoviridae, gênero Morbillivirus que, apesar de estar disponível uma vacina, segura e eficaz contra a doença, esta ainda constitui uma importante causa de morbidade e mortalidade infantil em muitos países em desenvolvimento. Embora exista apenas um tipo antigênico do vírus do sarampo, estudos de caracterização genética dos vírus de tipo selvagem permitiram a identificação oito subtipos (A-H) e 24 genótipos. O Brasil eliminou a transmissão autóctone do vírus do sarampo a partir do ano 2000. A partir de então foram confirmados vários casos de sarampo importados ou relacionados a importações, principalmente do genótipo D4. A epidemiologia molecular dos vírus do sarampo baseada nas análises da região C-terminal da nucleoproteína tem demonstrado uma diversidade limitada entre as cepas circulantes, dificultando dessa forma a determinação da origem dos vírus usando apenas essa região. O objetivo deste trabalho foi caracterizar geneticamente os genótipos D4 dos vírus do sarampo detectados no Brasil no período de 2003 a 2012, e para tal, as sequências da proteína H completa e do gene N parcial foram analisadas. Os casos positivos para o genótipo D4 foram previamente identificados pela amplificação dos 450nt da região C-terminal da proteína N por RT-PCR, as sequências completas do gene H destas amostras foram diretamente amplificadas por RT-PCR a partir das amostras clínicas e posteriormente sequenciado.As análises filogenéticas da sequência de nucleotídeos do gene N e do gene H completomos traram que os vírus do sarampo genótipo D4 detectados no Brasil podem ser resultado de várias importações de diferentes variantes do mesmo genótipo que circulam na Europa. Foram identificadas mutações nas sequências de aminoácidos tantodo gene N parcial como do gene H completo dos genótipos D4 dos vírus do sarampo detectados no Brasil...

Measles is a highly infectious viral exanthem of the family Paramyxoviridae, genusMorbillivirus. Despite the availability of a safe and effective vaccine, measles infectioncontinues to be an important cause of infantile morbidity and mortality in developingcountries. Although there is only one antigenic type of the measles virus, geneticcharacterisation of the wild-type virus identified 8 subtypes (A-H), with a total of 24genotypes being recognised. From 2000, the indigenous transmission of the measlesvirus has been eliminated in Brazil. Since then, several cases of imported measles, orcases associated with importations, were confirmed, principally of genotype D4. Themolecular epidemiology of the measles virus based on analyses of the C-terminal regionof the nucleoprotein has demonstrated a limited diversity between circulating strains,making it difficult to determine the origin of the virus by this genomic region alone. Theobjective of this study was to genetically characterise the D4 genotype of measlesviruses detected in Brazil from 2003 to 2012 by analysing sequences from the completeH gene and partial N gene of the virus. Cases positive for measles genotype D4 werepreviously identified by the amplification of a 450nt of the C-terminal region of the Nprotein by RT-PCR. The complete H gene from these samples was amplified by RTPCRdirectly from clinical samples and subsequently sequenced. Phylogenetic analysisof the nucleotide sequences of the N gene and the complete H gene demonstrated thatthe measles D4 genotype detected in Brazil may have been a result of severalimportations of different variants of the same genotype circulating in Europe...

Production of CCHF Virus-Like Particle by a Baculovirus-Insect Cell Expression System / 中国病毒学·英文版

Crimean-Congo Haemorrhagic Fever Virus(CCHFV)is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes,high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research,the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope,Virus-Like Particles (VLPs)with various size and morphology were observed in cytoplasmic vesicles in the infected cells.Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation,which was confirmed by Western blot analysis and immuno-electron microscopy(IEM).

Study on the changing regularity of special antibody and expression of stomach and enteric involvement on SARS-coronavirus infection in the recovery period of severe acute respiratory syndrome / 中华流行病学杂志

Objective To study the change of special antibodies titer IgG, IgM and nucleocaspid to SARS coronavirus (CoV) and observing the expression of stomach and enteric involvement on SARS-CoV infection by monoclonal antibody against N protein of SARS-CoV in the 7- year recovery period among family clustering cases of severe acute respiratory syndrome. Methods Special antibody titer to SARS-CoV of 14 patients from 5 different families and their 10 kinfolks continuously tested by IFA and antigen-capturing ELISA methods. Samples were taken in the 1st-7th year periods after SARS patients infected by SARS-CoV, being diluted and measured on it titers of three kinds of antibodies. Immunochemical staining with monoclonal antibody (mAb) against N protein of SARS-CoV was used to determine the stomach and enteric tissues among 5 SARS patients with their nucleocaspid antibody titer ascended obviously after 1st-7th year. Results When testing the IgG antibody titer of the 14 SARS patients by IFA method, the average titer was 1/71 (95%CI:1/58-1/85) in the 1st year, but began to descend in the following years, and the IgG antibody of the most SARS patients disappeared in the 7th year. Regarding the IgM titer, it disappeared in most of the SARS patients 1 year later. The average value of nucleocaspid antibody titer was 1/146 (95% CI:1/122-1/171) in the 1st year, and it descended as the IgG antibody titer did. In 5 cases, differences appeared.The nucleocaspid antibody titer was between 1/156 and 1/210 in 3 cases, and 2 cases were normal.Immunochemical staining with mAb against N protein of SARS-CoV was identified in the stomach and enteric tissues of 5 SARS patients with the nucleocaspid antibody titer increased significantly, 1st-7th year later. The five patients were detected by gastroscopy detection and cell immunohistochemistry test. 3 cases showed N protein antibody positive in the serum, and positive immunohistochemical expression in most of the cytoplasm in the gastric tissue mucous gland epithelial cells. 1 case also expressed in the intestinal tissue slurry columnar epithelium and interstitial cells. The other two cases showed negative on both serum N protein antibody and immunohistochemical expression. The biopsy results of the 5 patients were as follows: 1 case diagnosed as "signet-ring cell carcinoma of the stomach and rectum multiple transfer", 1 case of gastric polyp, 1 case of superficial antral gastritis and 2 cases were normal. Conclusion By testing the special IgG, IgM, nucleocaspid antibody to SARS-CoV of the 14 family clustering cases , we found that they all decreased in the 7th year, and most of them disappeared. The nucleocaspid antibody titer was related to pathogenetic condition. SARS-CoV was proved to be still present in stomach and enteric tissues of SARS patients with the nucleocaspid antibody titer increased significantly after the 7th year.

Development of Competitive ELISA for Detection of Avian Metapneumovirus Antibodies in Chicken

Avian metapneumovirus (aMPV) causes an acute and highly contagious upper respiratory tract infection in turkeys and chickens. In this study, a competitive ELISA (C-ELISA) was developed for the detection of antibodies to aMPV in chicken sera and/or their egg yolks. This assay is based on the competitive binding of monoclonal antibody with serum antibodies to recombinant aMPV N protein expressed by a recombinant baculovirus. The C-ELISA showed specificity and sensitivity of 100% and 98.0%, respectively, when compared to the virus neutralization test. In specific pathogen-free chickens experimentally infected with aMPV SC1509 strain, the C-ELISA started to detect antibodies to aMPV as early as 5 days post infection from birds infected with aMPV, while a commercial ELISA kit detected first 10 days post infection. The C-ELISA was similar or superior to a commercial ELISA kit when serum and egg yolk samples collected from chickens on six outbreak farms were tested for diagnosis. The C-ELISA developed in the present work provides a short turnaround time and can be a useful diagnostic and screening tool for aMPV infection in the field.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Application of Hantavirus N Protein Expressed by E. coli System

Hantaan viruses cause haemorrhagic fever with renal syndrome (HFRS), resulting in severe morbidity and mortality in humans. The genome of Hantaan virus is composed of three segmented and single stranded negative sense RNA genome. In this study, we expressed nucleocapsid (N) proteins of Hantaan 76-118, Seoul 80-39 and Hantaan virus isolated in Korea (01-23) using E. coli system. These N proteins were fused with a thioredoxin protein for secretion of the expressed protein. The antigenicity of each expressed N proteins was examined in Western blot with sera from HFRS patients and normal controls. The expressed N proteins did not show any cross-reactivity with sera obtained from patients with leptospirosis and tsutsugamushi disease. These results suggest that our recombinant N proteins can be used for the development of diagnostic system to distinguish between HFRS and leptospirosis or tsutsugamushi.

Characterization of Antigenic Sites on the Rinderpest Virus N Protein Uusing Monoclonal Antibodies

The N protein of the rinderpest virus (RPV) was analyzed topologically and antigenically by using anti-N monoclonal antibodies (Mabs). Ten Mabs were raised against the N protein of the RPV. At least six non-overlapping antigenic sites (sites A-F) were delineated by competitive binding assays using biotinylated Mabs. Of them 5 sites (A, C, D, E and F) on the N protein were recognized by RPV-specific Mabs in ELISA and IFA while site B was recognized by Mabs reacting with both RPV and PPRV. Non- reciprocal competition was found among sites C, D and E. Recombinant RPV N protein after exposure to 0.2% SDS exhibited higher ELISA titers in all Mabs recognizing 6 sites. Four sites (A, B, E and F) on 2% SDS-treated N protein lost completely reactivity with Mabs while the remaining sites (C and D) on the protein retained their antigenicity to some degree. It indicates that two sites (C and D) were sequential. Six representative Mabs bound to each site exhibited competition with rinderpest antibodies in a blocking ELISA, indicating that the sites were actively involved in antigenicity in cattle.

Preparation and characterization of polyclonal antibody against N protein of SARS-CoV / 解放军医学杂志

Objective To prepare and characterize the polyclonal antibody against N protein of SARS-CoV. Methods The polyclonal antibody against SARS-CoV N was obtained from immunized rabbit with purified GST-N. The titer of the antibody was determined by indirect ELISA, and the specificity by Western blot and immunochemical staining. Results The rabbit′s antibody against SARS-CoV N was prepared successfully. The titer of antiserum against SARS-CoV N was about 1.2?10~ -5 . Western blot and immunochemical staining analysis showed that the polyclonal antibody could bind to the expressed fusion protein specifically. Conclusion The rabbit′s antibody against SARS-CoV N has been prepared successfully, and it can be a useful reagent for clinical diagnosis and further research.

The expression and activity determination of N protein of Sars-CoV / 解放军医学杂志

Objective To construct an expression vector pGEX-2T/N, and to express the fusion protein consisting of N protein of SARS-CoV in E. coli.Methods The N region gene of SARS-CoV was obtained by RT-PCR. The expression vector PGEX-2T/N was constructed by DNA recombination. The recombinant plasmid was transformed into E. coli BL21(DE3). The expression of the fusion protein was determined by Western blot with anti-SARS-CoV antibody positive blood sera. Results The N region gene of SARS-CoV was obtained. The fusion protein GST-N was soluble. Western blot analysis showed that the reaction of GST-N to anti-SARS-CoV sera was positive. Conclusion The pGEX-2T/N has been constructed and expressed in the form of fusion protein GST-N successfully, and the result lays the foundation for further study of SARS-CoV N protein.

Construction of PGEFP-C1/N and its localization in transfected cell / 解放军医学杂志

Objective To identify the subcellular location of S AR S-CoV N protein in mammalian cells. Methods The gene fraction of SARS-CoV was cloned into the PGEFP-C1 plasmid to construct expression vecto r PGEFP-C1/N. The subcellular location of N in A549 and VeroE6 cells was observ ed under fluorescence microscope with the aid of transient transfection techniqu e. The expression of the fusion protein (GFP-N) was detected by Western blot. Results The PGEFP-C1/N was constructed. N protein was localiz ed in the cytoplasm of transfected cells and detected by Western blot. C onclusion N protein was localized in the cytoplasm of mammalian cells.

Expression,purification and antigen analysis of SARS-associated coronavirus nucleocapsid protein in Pichia pastoris system / 第三军医大学学报

Objective To study the expression of SARS-associated coronavirus nucleocapsid(N) gene in Pichia pastoris and to obtain recombinant N protein with good biological activity.Methods The gene encoding the full N protein of SARS-CoV was amplified by PCR and cloned into Pichia pastoris expression vector pPICZA.The recombinant expression plasmid pPICZA/N was transformed into X-33,KM71H and GS115. The positive insert transformants were screened,cultured and induced by methanol.The recombinant protein was further purified with Ni affinity chromatography.Antigen activity was detected with anti-N monoclonal antibody,polyclonal antibody and sera from SRAS patients.Results SDS-PAGE and immunological analysis demonstrated that only Pichia pastoris transformants KM71H/pPICZA/N and X-33/pPICZA/N expressed Mr 70 000 fusion protein with special antigenicity.Conclusion SARS-CoV N protein expression in Pichia pastoris has been achieved,establishing the basis for further study of biological and immunological function of N antigen.