Résumé
Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate. Materials and Methods: B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Results: The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high- frequency distribution was improved to 85%. Expression of recombinant protein in E. coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000. Conclusion: According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.
Résumé
Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Material(s) and Method(s): B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Result(s): The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high-frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion(s): According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.
Résumé
Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Material(s) and Method(s): B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Result(s): The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high-frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion(s): According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.
Résumé
Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Material(s) and Method(s): B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Result(s): The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high-frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion(s): According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.
Résumé
Background: Post-COVID-19 conditions are defined as new, recurring, or ongoing health issues which present weeks after SARS-CoV-2 infection. The gastrointestinal (GI) involvement of COVID-19 suggests that a group of patients with lingering GI symptoms may develop Post-COVID-19 DGBI including irritable bowel syndrome (IBS) (Schmulson M et al. Am J Gastroenterol. 2021;116:4-7). In this study, we aimed to determine the epidemiological features of Post-COVID-19 DGBI. Methods: Subjects with confirmed COVID-19 at least 6 months before the study who had sustained GI symptoms were invited to complete an internet-based survey on Qualtrics, between March and August 2021. The survey included demographics, acute symptoms, comorbidities, as well as Rome IV questionnaire, Generalized Anxiety Disorder questionnaire (GAD-7) and Patient Health Questionnaire (PHQ)-9 for depression. Data was analyzed using ANOVA and multivariate analysis. Findings were reported as percentage or [p-value;(95% odds ratio CI)]. Results: Overall, 164 subjects (70% female, 14% male, and others unknown) with a positive COVID-19 test completed the survey. Among them, 4% were >65 years old and 24% reported hospitalization. Body mass index ³30 was present in 38%, diabetes in 6.7%, and vitamin D deficiency in 11% of the participants. In total, 108 (66%) subjects fulfilled Rome IV criteria for at least one DGBI. Of 108 with DGBI, only 27 (25%) had DGBI before COVID-19;DGBI developed in 81 subjects after COVID-19. The most common Post-COVID-19 DGBI were functional dyspepsia observed in 38 (postprandial distress syndrome n=31, epigastric pain syndrome n=22) followed by IBS in 26 subjects (IBS with Diarrhea n=7, IBS with Constipation n=4, Mixed-IBS n=14, Unsubtyped IBS n=1) (Table-1). The risk factors of severe COVID-19 including age >65, diabetes, and obesity were not associated with developing Post-COVID- 19 DGBI. Seventy (86%) of subjects with Post-COVID-19 DGBI had at least one GI symptom (abdominal pain, nausea/vomiting, and/or diarrhea) in the acute phase of COVID-19. Nausea/ vomiting during the acute illness increased [p-value of 0.02 with 95% OR CI (0.7-10.4)], and BMI less than 25 also increased the odds [p-value of 0.03 (95% OR CI: 0.26-8.4)] for Post-COVID-19 IBS. Anxiety was present in 48% and depression in 65% of subjects with Post-COVID-19 DGBI. Conclusions: Post-COVID-19 DGBI are new entities associated with a high rate of anxiety and depression. Although the majority of those with Post-COVID-19 DGBI reported having GI symptoms in the acute illness, some appeared in subjects without acute GI symptoms. (Table Presented)