Expression and receptor binding characteristics of porcine P6 genotype rotavirus GST-VP8*-Z84 protein / 中华实验和临床病毒学杂志

Objective@#The gene engineering technique was used to express the P[6] genotype rotavirus (rotaviruses, RVs) GST-VP8*-Z84 protein from pigs, and the binding characteristics of the protein to oligosaccharide and salivary receptor were studied.@*Methods@#The GST-VP8*-Z84 protein was purified by GST Escherichia coli expression system and affinity chromatography using porcine P[6]. Enzyme-linked immunosorbent assay (ELISA) saliva binding test and oligosaccharide binding test were used to analyze the binding characteristics of the genotype to receptors.@*Results@#Porcine P[6] GST-VP8*-Z84 protein bound well to mucin core 2.@*Conclusions@#The potential receptor of P[6] RV may be the core of mucin, which may provide the experimental basis and theoretical basis for the mechanism of rotavirus and receptor interaction and the development of RV vaccine and highly effective therapeutic drugs.

The expression and purification of VP8* core protein of the rotavirus P 6 Genotype LL4260 / 中华实验和临床病毒学杂志

Objective@#The VP8* core protein of rotavirus P[6] genotype LL4260 was purified by prokaryotic expression, which is important for further study of protein structure and function.@*Methods@#The P[6] genotype LL4260 strain was obtained by PCR.The recombinant plasmid pET30 a-LL4260VP8*core was inserted into pET30 a vector and transformed into BL21 (DE3) competent cells with the correct recombinant plasmid. The expressed protein is purified by affinity chromatography and molecular sieve chromatography.@*Results@#The pET30 a-LL4260VP8* core region protein is soluble in the supernatant and proteins of approximately 22 kDa are identified by electrophoresis using purified proteins.@*Conclusions@#In this study, LL4260 containing pET30 a-LL4260VP8* core plasmid was successfully constructed and LL4260 strain VP8* protein was expressed.

Protein expression and receptor binding specificity of porcine P19 rotavirus VP8* / 中华实验和临床病毒学杂志

Objective@#To explore the receptor binding specificity of VP8* protein of porcine P[19] rotaviruses (RVs) with oligosaccharides.@*Methods@#The porcine P[19] VP8* protein was expressed and purified. The receptor binding specificity of P[19] VP8* was analyzed by oligosaccharide binding and saliva binding assay.@*Results@#The P[19] VP8* protein showed significant binding to mucin cores, especially mucin core 2.@*Conclusions@#Mucin core 2 may be a potential receptor for the porcine P[19] RV, which provides certain basis for the study of virus infection mechanism and RV surveillance.

Preparation and characterization of an egg yolk antibody (IgY) against the recombinant human A rotavirus VP8 / 中华微生物学和免疫学杂志

Objective To prepare an egg yolk antibody ( IgY) against the recombinant human A rotavirus VP8 and evaluate its biological characteristics .Methods The complete VP4 gene was used as the template to clone VP8 DNA fragment by PCR .The VP8 gene was then cloned into the vector pET 28a for ex-pression of VP8 protein in the prokaryotic system.The expression plasmid of pET28a-VP8,identified by DNA sequencing,was transformed into E.coli BL21 to induce the expression of protein by 0.5 mmol/L IPTG at 30℃.The purified and refolded recombinant VP 8 protein was used to immunize laying hens in combination with complete Freund′s adjuvant ( CFA ) .Anti-VP8 IgY antibodies were extracted from egg yolks by using water dilution-ultrafiltration assay and were further analyzed by SDS-PAGE,ELISA,Western blot and Dot blot assay,respectively.Results The recombinant VP8 protein was expressed in E.coli BL21 as an inclusion body and its molecular weight was about 35 ×10 3 .The VP8 protein was well refolded in a buffer containing Tris-HCl (pH8.0).The isolated anti-VP8 IgY antibodies showed a high titer of 1∶100 000 and a high stabil-ity under the condition of pH3-11 and temperature 25-65℃.Moreover,the anti-VP8 IgY antibodies specific-ally recognized the wild type human A rotavirus .Conclusion The prepared anti-VP8 IgY antibodies showed the advantages of high titer ,good specificity and stability .It might be used as the tool in further investigation for the prevention and treatment of diarrhea caused by human A rotavirus .

Expresión y purificación de las proteínas estructurales del rotavirus VP5* y VP8* en bacterias E. coli BL21(DE3) / Expression and purification of rotavirus structural proteins VP5* and VP8* in bacteria E. coli BL21(DE3)

La caracterización de las proteínas estructurales del rotavirus y de las proteínas de la superficie de la célula hospedera implicadas en la unión y penetración del virion requiere de la disponibilidad de cantidades suficientes y de alto grado de pureza de estas proteínas. Por lo tanto, el objetivo de este trabajo fue expresar y purificar las proteínas estructurales del rotavirus de la cepa RRV, VP5* y VP8*, y producir anticuerpos policlonales dirigidos contra ellas. Se expresaron las proteínas recombinantes VP5* (rVP5*) y VP8* (rVP8*) en bacterias E. coli BL21(DE3) transfectadas con el plásmido pGEX-4T que contenía sus secuencias codificantes. Se consideraron como variables el medio de crecimiento, número de bacterias antes de inducir la expresión, concentración del inductor y tiempo de inducción. La mayor proporción de rVP8* se obtuvo cuando las bacterias transformadas se cultivaron en medio LB y la inducción se llevó a cabo con 1 mM de IPTG cuando el cultivo alcanzó una OD 600 nm de 0.5 y la inducción se mantuvo durante 6 h. rVP5* alcanzó la mayor proporción cuando células a una OD 600 nm de 0.2 fueron inducidas con 0.5 mM de IPTG durante 4 h en medio 2XYT en presencia de glucosa al 2 %. Las proteínas recombinantes obtenidas, acumuladas en la fracción insoluble, fueron solubilizadas con detergentes iónicos y no iónicos, seguido de purificación mediante cromatografía de afinidad antes de ser empleadas como antígenos para la producción de anticuerpos policlonales en conejos. Estos anticuerpos se caracterizaron mediante su capacidad de reconocimiento de los antígenos correspondientes en ELISA, "Western blotting", y en ensayos de inmunocitoquímica en células infectadas con rotavirus RRV. La cantidad y el grado de pureza de las proteínas recombinantes obtenidas, y los anticuerpos dirigidos contra ellas, anticipan su utilidad como herramientas en la caracterización de la interacción virus-célula.

The characterization of rotavirus structural proteins and the cell surface proteins involved in virion binding and penetration depends on the availability of substantial amounts of highly purified proteins. The aim of the present work was to express and purify the rotavirus structural proteins VP5* and VP8* in order to produce polyclonal antibodies against them. Recombinant proteins VP5* (rVP5*) and VP8* (rVP8*) were expressed in E. coli cells transfected with pGEX-4T or pET 28a containing their corresponding encoding sequences. Culture medium, bacterial concentration before induction, inductor concentration and induction time were used as variables. The greater proportion of rVP8* was obtained when transfected bacteria were grown in medium LB and induction was started by adding 1 mM IPTG to cells at OD 600 nm 0.5 followed by 6 h-induction. The highest proportion of rVP5* was reached when cells at OD 600 nm 0.2 were induced with 0.5 mM IPTG for 4 h in medium 2XYT containing 2% glucose. The recombinant proteins accumulated in the insoluble fraction were solubilized with ionic and non-ionic detergents, and purified by affinity chromatography before being used as antigens for production of rabbit polyclonal antibodies. The antibodies produced were characterized through their ability to recognize the corresponding antigens in ELISA, Western blotting, and immunochemistry assays in rotavirus infected cells. The amount and purity of recombinant proteins obtained in this work, and the antibodies against them, are expected to be useful for the characterization of the virus-cell interaction.