No abstract

Study on sialidases as antiviral agents has been widely performed, but many types of sialidase had not been tested for their antiviral activity. One of such sialidase is the NanB sialidase of Pasteurella multocida, which has never been isolated for further study. In this study, the activity of NanB sialidase was investigated in silico by docking the NanB sialidase of Pasteurella multocida to the Neu5Acα(2-6)Gal ligand. Additionally, some local isolates of Pasteurella multocida, which had the NanB gene were screened, and the proteins were isolated for further testing regarding their activity in hydrolyzing Neu5Acα(2-6)Gal. In silico studies showed that the NanB sialidase possesses an exceptional affinity towards forming a protein-ligand complex with Neu5Acα(2-6)Gal. This was further confirmed by showing that a dose of 0.258 U/ml (100%) NanB sialidase of Pasteurella multocida B018 can hydrolyze up to 44.28% of Neu5Acα(2-6)Gal in chicken red blood cells and 81.95% in rabbit red blood cells. This study suggested that the NanB sialidase of Pasteurella multocida B018 has a potent antiviral activity that can inhibit avian influenza virus infection.

ABSTRACT Covid-19 infection is still a health problem in Indonesia and even throughout the globe. To control Covid-19, efforts are made for each country to conduct research to develop raw materials that can be used for anti-SARS-CoV-2 detection in serological diagnostics. This study aimed to construct recombinant plasmids expressing SARS-CoV-2 antigen so that it be used in the production of raw materials in developing the SARS-CoV-2 serological test. The genes coding spike and nucleocapsid recombinant antigens cloned in pUC57 were subcloned into the pQE80L expression vector to produce pQE80L-spike and pQE80L-nucleocapsid plasmids. The recombinant bacterial selection was carried out by colony Polymerase Chain Reaction, recombinant plasmids were analyzed with BamHI and HindIII restriction enzymes. The recombinant plasmids were verified by sequencing. The PCR results showed the colonies containing recombinant plasmids pQE80L-spike and pQE80L-nucleocapsid produced deoxyribonucleic acid bands of 1438 bp and 772 bp, respectively. The restriction analysis of pQE80L-Spike and pQE80L-nucleocapsid plasmids produced 4709 bp vector fragments and 1188 bp and 522 bp of inserted DNA, respectively. The sequencing showed the spike and nucleocapsid coding gene have been cloned into pQE80L. The construction of SARS-CoV-2 plasmid expression spike and nucleoprotein SARS-CoV-2 was successful to develop qualitative and quantitative anti-SARS-CoV-2 antibody detection tests.   Keywords: spike, nucleocapsid, SARS-CoV-2, cloning, anti-SARS-CoV-2 detection   ABSTRAK Infeksi Covid-19 masih menjadi masalah kesehatan di Indonesia bahkan juga di seluruh negara. Dalam rangka pengendalian infeksi Covid-19, maka setiap negara berupaya melakukan penelitian untuk mengembangkan bahan baku yang dapat digunakan untuk uji deteksi antibodi anti-SARS-CoV-2, salah satunya adalah diagnostik serologi. Tujuan penelitian ini adalah mengkonstruksi plasmid rekombinan pengekspresi antigen SARS-CoV-2 sehingga dapat digunakan dalam memproduksi bahan baku dalam pengembangkan uji diagnostik serologi SARS-CoV-2. Gen pengekspresi antigen rekombinan spike (S) dan nukleokapsid (N) yang berada dalam pUC57 disubklona ke dalam vektor ekspresi pQE80L untuk menghasilkan plasmid pQE80L-Spike dan pQE80L-Nukleokapsid. Seleksi bakteri rekombinan dilakukan dengan Polymerase Chain Reaction (PCR) koloni, plasmid rekombinan dianalisis dengan enzim restriksi BamHI dan HindIII, serta diverifikasi dengan sekuensing. Hasil PCR menunjukkan koloni mengandung plasmid rekombinan pQE80L-Spike dan pQE80L-Nukleokapsid menghasilkan pita deoxyribonucleic acid (DNA) masing-masing berukuran 1438 pb dan 772 pb. Analisis restriksi plasmid pQE80L-Spike dan pQE80L-Nukleokapsid menghasilkan fragmen vektor 4709 pb dan DNA sisipan berturut turut 1188 pb dan 522 pb. Hasil sekuensing menunjukkan spike dan nukelokapsid terklona dalam vektor pQE80L. Konstruksi plasmid pengekspresi spike dan nukleokapsid SARS-CoV-2 telah berhasil dilakukan untuk mengembangkan uji deteksi antibodi anti-SARS–CoV-2 baik kualitatif maupun kuantitatif.   Kata kunci: spike, nukleokapsid, SARS-CoV-2, kloning, deteksi antibodi anti-SARS-CoV-2

Background: High mutation rates in HIV-1 could affect the accuracy of diagnostic tests. Therefore, recombinant antigen that has an immunodominant and conserved region from HIV-1 need to be developed to detect HIV-1 infection in Indonesia.Materials and methods: The recombinant antigens comprise of Gag (p24), Pol and Env (gp41). Each antigens was expressed in the Escherichia coli expression system and purified using Ni-NTA chromatography. The reactivity of purified antigen against HIV antibodies was tested against a group of 50 HIV-positive plasma samples and 45 HIV-negative plasma samples in a recombinant immunoblot assay (RIBA) platform test. Moreover, 21 of 50 HIV-positive samples and 3 of 45 HIV-negative samples were also tested using HIV blot 2.2 to compare RIBA with a commercial western blot kit. Ten HBV-positive and 10 HCV-positive plasma samples were used to check cross-reactivity with HIV recombinant proteins in RIBA.Results: All HIV-positive samples (100%) tested with RIBA were reactive towards Gag (p24), Pol, Env (gp41). Otherwise, 3 of 21 HIV-positive samples assayed with HIV blot 2.2 were not reactive to Pol protein. All HIV-negative samples tested with RIBA and 3 HIV-negative samples tested with HIV blot 2.2 did not produce any bands of HIV antigens. Few HBV and HCV samples showed reactivity towards HIV recombinant proteins.Conclusion: Each recombinant protein, Gag (p24), Pol, Env (gp41), could be expressed and purified, as well as had reactivity to HIV-positive samples in RIBA test. Therefore, RIBA can be used as a diagnostic test to detect HIV-1 infection in Indonesia.Keywords: diagnostic, HIV-1, immunodominant, recombinant immunoblot assay (RIBA)