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"Expression and reactivity of spike and nucleocapsid recombinant antigens for detection of anti-SARs C0V-2 antibodies

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Expression and reactivity of spike and nucleocapsid recombinant antigens for detection of anti-SARs C0V-2 antibodies"

"SARS-CoV-2 merupakan penyebab COVID-19 yang melanda dunia sejak akhir 2019. Virus ini telah menyebar secara luas di dunia akibat infektifitasnya yang tinggi. Salah satu penyebab tingginya infektifitas virus ini adalah spike glycoprotein. Spike glycoprotein merupakan salah satu protein yang terdapat pada SARS-CoV-2. Spike glycoprotein berperan secara langsung alam mekanisme infeksi dengan cara membentuk ikatan dengan reseptor ACE-2 pada sel inang. Inhibisi spike glycoprotein dapat menjadi salah satu cara pengobatan COVID-19. Dalam penelitian ini, antivirus yang sudah dipasarkan sebagai basis data akan di-repurpose menjadi antivirus SARS-CoV-2, kemudian dilakukan modifikasi terhadap senyawa yang terpilih menjadi senyawa organoselen. Penelitian dilakukan dengan cara in silico. Untuk simulasi molecular docking, digunakan software MOE2014.09 untuk mendapatkan informasi tentang interaksi antara spike glycoprotein dengan ligan, baik antivirus maupun antivirus hasil modifikasi. Melalui analisa nilai energi pengikatan dan uji farmakologi, diperoleh 3 ligan terbaik dari antivirus (Ombitasvir, Elbasvir, dan Ledipasvir) serta antivirus modifikasi (ModL1, ModL2, dan ModL6).

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SARS-CoV-2 is the cause of COVID-19 that has hit the world since the end of 2019. This virus has spread widely in the world due to its high infection. One of the causes of the high infectivity of this virus is the spike glycoprotein. The spike glycoprotein is a protein found in SARS-CoV-2. The glycoprotein spike directly plays a role in the infection mechanism by forming a bond with the ACE-2 receptor on the host cell. Inhibition of spike glycoprotein can be one way of treating COVID-19. In this study, the antivirals that have been marketed as databases will be repurposed into SARS-CoV-2 antivirals, then the selected compounds will be modified into organoselenium compounds. The research was conducted through in silico. The molecular docking simulation was conducted by using MOE2014.09 to retrieve information about the interaction between the protein-ligand from unmodified antivirus as well as modified antivirus. Through the binding energy value and pharmacological tests, the three best ligands are obtained from the unmodified antivirus (Ombitasvir, Elbasvir, and Ledipasvir) and the modified antivirus (ModL1, ModL2, and ModL6)."

"Latar belakang: Dari 36,9 juta orang yang terinfeksi oleh Human Immunodeficiency Virus (HIV) pada akhir tahun 2018 di seluruh dunia, terdapat sekitar 1-2 juta yang terinfeksi HIV-2. Meskipun HIV-2 kurang patogen dibandingkan dengan HIV-1, misdiagnosis infeksi HIV-2 dapat menyebabkan kegagalan pengobatan yang berujung pada perkembangan Acquired Immune Deficiency Syndrome (AIDS). Diagnostik yang akurat diperlukan untuk menentukan apakah suatu individu telah terinfeksi HIV-1, HIV-2 atau koinfeksi HIV-1 dan HIV-2. Metode: Penelitian ini menggunakan DNA sintetik penyandi antigen rekombinan gp125-gp36 HIV-2 yang bersifat immunodominan, lestari, telah dioptimasi kodon untuk sistem ekspresi E. coli, dan telah dianalisis struktur sekunder mRNAnya. DNA sintetik diklona ke plasmid pQE80L untuk diekspresikan, kemudian dipurifikasi menggunakan kromatografi afinitas Ni-NTA. Antigen rekombinan kemudian diuji reaktivitasnya dengan antibodi anti-HIV-2, serta 7 serum positif HIV-1, HBV, HCV, dan serum normal.Hasil: Gen sintetik berhasil dikonstruksi pada plasmid pQE80L dan dapat diekspresikan dengan induksi 0,1 mM IPTG selama 4 jam. Antigen rekombinan terpurifikasi secra optimal pada kondisi denature dengan pH elusi 4,5. Selanjutnya, hasil uji reaktivitas menunjukkan hasil reaktif untuk antibodi anti HIV-2 dan tidak tidak reaktif untuk 7 sampel serum positif HBV, HCV, dan serum normal. Sedangkan untuk serum positif HV-1, terdapat hasil reaktif pada sampel serum nomor 3 yang diduga disebabkan oleh protein kontaminan dari E. coli.Kesimpulan: Antigen rekombinan gp125-gp36 HIV-2 untuk deteksi antibodi anti-HIV-2 telah berhasil dikembangkan, akan tetapi perlu dilakukan optimasi lebih lanjut untuk mendapatkan antigen rekombinan yang benar-benar murni.

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Background: From 36.9 million people worldwide infected by the Human Immunodeficiency Virus (HIV) at the end of 2018, 1-2 million are infected by HIV-2. Although HIV-2 is less patogenic than HIV-1, misdiagnostic of HIV-2 infection could effect to treatment failure which leads to development of Acquired Immune Deficiency Syndrome (AIDS). Accurately diagnostic is required to ascertain whether an individual has been infected HIV-1, HIV-2, or HIV-1 and HIV-2 co-infection.

Method: This study used immunodominant and sustainable synthetic DNA encoding of recombinant antigen gp125-gp36 HIV-2 which was optimized by codon for E. coli expression system, and analyzed the secondary structure of its mRNA. Synthetic DNA was cloned to the pQE80L plasmid to be expressed, purrifyed using Ni-NTA affinity chromatography. Recombinant antigen therefore was verified for reactivity by anti-HIV-2 antibodies and 7 positive serum of HIV-1, HBC, HCV, and normal serum.

Result: The synthetic gene was succesfully constructed on pQE80L plasmid and able to be expressed by induction of 0.1 mM IPTG for 4 hours. Recombinant antigen was optimally purified in denature conditions due to elusion pH of 4.5. Furthermore, the reactivity test revealed reactive result for anti HIV-2 antibodies and unreactive to 7 positive sample serum of HBC, HCV, and normal serum. While positive serum HIV-1 demonstrate a reactive result in sample serum number 3 supposed causing by protein contaminants from E. Coli. Conclusion: Recombinant antigen gp125-gp36 HIV-2 for the detection anti HIV-2 antibodies has been succesfully developed, however further optimization is required in case to obtain truly pure recombinant antigens."

"ABSTRAKHepatitis C virus HCV menginfeksi lebih dari 170 juta penduduk dunia dan menyebabkan penyakit hati kronis yang berkembang menjadi sirosis dan kanker hati. Diagnosis yang akurat sangat diperlukan untuk memberikan penanganan tepat secara dini, termasuk mencegah penularan virus tersebut secara lebih luas. Pada penelitian ini plasmid pQE80L-HCV_ME telah berhasil dibuat untuk produksi antigen rekombinan HCV. Gen pengkode antigen tersebut dirancang berdasarkan multiepitop yang bersifat imunodominan, lestari, mewakili subtipe HCV di Indonesia dan global. Gen tersebut dibuat dengan teknik DNA sintetik kemudian diklona dari plasmid pUC57 ke pQE80L. Pengklonaan dilakukan menggunakan situs restriksi BamHI dan HindIII dalam sel E. coli Top10. Plasmid pQE80L-HCV_ME kemudian diverifikasi dengan PCR koloni, analisis restriksi, dan sekuensing.

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ABSTRACTHepatitis C virus HCV have been infected more than 170 million people in the world and caused chronic liver disease that lead to liver cirrhosis and hepatocellular carcinoma. Accurate diagnosis is very important to give early proper treatment and to prevent HCV transmission broadly. In this research pQE80L HCV ME plasmid has been successfully created to produce HCV recombinant antigen. Gene that encodes antigen was designed based on multiepitop sequences from immunodominat region, conserve, and represent the most prevalence HCV subtypes in Indonesia and global. The gene was generated through synthetic DNA then be cloned from pUC57 plasmid to pQE80L. Cloning was performed by using BamHI dan HindIII in E. coli Top10 cell. pQE80L HCV ME plasmid then be verified by colonies PCR, restriction analysis, and sequencing."

"The objective were to know the base medium, growth regulator concentration of Kinetin, and combination of this treatment that had the best response to rooting phase of Sandalwood. This study was expected to play role in contributing great advantages to support the plant material provision in operational scale.Generally, the protocol of tissue culture of Sandalwood had been acknowledged, however there were still problems on rooting phase. Therefore the study wasfocused on 1/2 MS medium application, 1/2 GD, and 1/2 WPM, also application of Kinetin in different levels of concentration (0; 0,25; 0,50; 0,75; and 1 mg/1) on root development in Sandalwood.Study result concluded that the base medium of 1/2 MS and application of Plant Growth Regulators IBA 20 mg/1 combined with IAA 1 mg/1, and treatment of 0,75 mg/1 Kinetin concentration had the best response to growth and enlargement of Sandalwood root."

"Salah satu terapi COVID-19 adalah plasma konvalesen yang disiapkan Unit Transfusi Darah dari donor yang telah sembuh dari COVID-19. Plasma konvalesen mengandung antibodi netralisasi yang menghambat interaksi antara protein S dengan reseptor ACE2 dengan persyaratan minimal titer 1:160 sehingga diperlukan sistem deteksi antibodi netralisasi seperti tes serologi berbasis ELISA kompetitif yang mudah, murah, cepat dan tidak membutuhkan BSL 3 atau 2. Uji ini membutuhkan protein rekombinan spike S1 yang dapat diekspresikan pada sistem ekspresi mamalia. Penelitian ini bertujuan untuk mendeteksi antibodi spesifik SARS-CoV-2 pada plasma konvalesen COVID-19 menggunakan protein rekombinan Spike S1.Penelitian ini menggunakan plasmid pD609 sebagai vektor ekspresi yang terdapat gen spike S1. DNA ditransfeksi secara transien ke sel CHO. Immunostaining dilakukan setelah transfeksi untuk melihat ekspresi protein rekombinan spike S1 pada sel CHO. Supernatan media sel CHO post transfeksi dianalisis dengan western blot dan ELISA untuk melihat reaktifitas terhadap serum konvalesen COVID-19. Hasil immunostaining menunjukkan plasmid pD609 S1 Spike Foldon-His dapat mengekspresikan protein rekombinan spike S1 SARS-CoV-2 pada sel CHO. Hasil Western Blot dan ELISA menunjukkan supernatan media sel kultur CHO post transfeksi reaktif terhadap serum konvalesen COVID-19. Protein rekombinan spike S1 memiliki potensi untuk dikembangkan dan digunakan dalam uji antibodi spesifik namun hasil ekspresi protein masih rendah.

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One of the therapies for COVID-19 is convalescent plasma prepared by the Blood Transfusion Unit from donors who have recovered from COVID-19. Convalescent plasma contains neutralizing antibodies that inhibit the interaction between S protein and ACE2 receptors with a minimum requirement of a titer of 1:160 so that a neutalizing antibody detection system is needed such as a competitive ELISA-based serological test that is easy, inexpensive, fast, and does not require BSL 3 or 2. S1 spike recombinant protein that can be expressed in mammalian expression systems. This study aims to detect SARS-CoV-2 specific antibodies in COVID-19 convalescent plasma using recombinant Spike S1 protein. This study used the pD609 plasmid as an expression vector containing the spike S1 gene. DNA was transiently transfected into CHO cells. Immunostaining was performed after transfection to see the expression of the S1 spike recombinant protein in CHO cells. The post-transfected CHO cell media supernatans were analyzed by western blot and ELISA to see the reactivity to COVID19 convalescent serum. Immunostaining results showed that the plasmid pD609 S1 Spike Foldon-His could express the SARS-CoV-2 spike S1 recombinant protein in CHO cells. The results of Western blot and ELISA showed that the post-transfection CHO cell culture media supernatant was reactive to COVID-19 convalescent serum. S1 spike recombinant protein has the potential to be developed and used in specific antibody assays, but the results of protein expression is still low."

"Penerapan antibodi monoklonal (mAb) anti-spike untuk digunakan dalam diagnosis SARS-CoV-2 memerlukan suatu proses purifikasi untuk mendapatkan suatu antibodi yang murni dan homogen sehingga dapat mendeteksi suatu patogen spesifik secara optimal. Penelitian ini bertujuan untuk memurnikan mAb terhadap protein spike SARS-CoV-2 dan membandingkan hasil purifikasi terbaik dari metode kromatografi afinitas dengan protein G dan kromatografi penukar ion sehingga diperoleh metode yang paling optimal dalam purifikasi mAb terhadap protein spike SARS-CoV-2. Purifikasi mAb anti-spike SARS-CoV-2 dilakukan menggunakan kromatografi afinitas dengan protein G dan kromatografi penukar ion. Hasil purifikasi dari kedua metode kromatografi dikarakterisasi dan diuji fungsionalitasnya menggunakan SDS-PAGE, pengukuran konsentrasi protein, ELISA indirect, dan western blot (WB). Hasil profil SDS-PAGE menunjukkan mAb hasil purifikasi menggunakan protein G pada fraksi 19GD dan 20GD memiliki profil pita protein dengan dua pita, yaitu heavy chain ~50 kDa dan light chain ~25 kDa dengan tingkat kemurnian mencapai 96%. Uji fungsionalitas dengan ELISA indirect menunjukkan fraksi 19GD dan 20GD memiliki nilai absorbansi sebesar 1,015 dan 1,021. Uji fungsionalitas dengan WB menunjukkan adanya pengikatan mAb fraksi 19GD terhadap protein RBD pada ukuran ~38 kDa. Hasil karakterisasi dan uji fungsionalitas mAb fraksi hasil purifikasi dengan resin penukar ion menunjukkan profil pita protein kontaminan, nilai absorbansi dari 0,49—0,82 , dan tidak terbentuk pita protein pada uji WB. Berdasarkan hasil tersebut, mAb anti-spike SARS-CoV-2 berhasil dimurnikan menggunakan kromatografi afinitas dengan protein G secara optimal.

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The application of anti-spike monoclonal antibody (mAb) for use in the diagnosis of SARS-CoV-2 requires a purification process to obtain a pure and homogeneous antibody so that it can detect a specific pathogen optimally. This research aims to purify anti-spike SARS-CoV-2 mAb and compare the best purification results from affinity chromatography with protein G and ion-exchange chromatography methods in order to obtain the most optimal method of purification of anti-spike SARS-CoV-2 mAb. Purification of anti-spike SARS-CoV-2 mAb was carried out using affinity chromatography with protein G and ion exchange chromatography. The purification results from both chromatographic methods were characterized and tested for functionality using SDS-PAGE, measurement of protein concentration, indirect ELISA, and western blot (WB). The results of SDS-PAGE profile showed that mAb purified using protein G in the 19GD and 20GD fractions had a protein band profile with two bands, namely heavy chain ~50 kDa and light chain ~25 kDa with a purity level of 96%. The functionality test with indirect ELISA showed that 19GD and 20GD fractions had OD values ​​of 1.015 and 1.021. Functionality test with WB showed the binding of mAb fraction 19GD to RBD protein at ~38 kDa. The results of characterization and functionality test of purified mAb fraction with ion exchange resin showed a contaminant protein band profile, absorbance values ​​from 0.49—0.82, and no protein band was formed in the WB test. Based on these results, anti-spike SARS-CoV-2 mAb was successfully purified using affinity chromatography with protein G optimally."

"Latar BelakangSejak awal pandemi, dinamika mutasi pada domain RBD pada protein S-glycoprotein SARS-CoV-2 telah mengubah patogenisitas varian yang beredar di Indonesia. Penelitian ini akan menganalisis tren mutasi pada berbagai sampel domain RBD di Indonesia yang telah dipublikasikan di Genomic Database dengan menggunakan genomic profilling MetodePasien yang terinfeksi COVID-19 di Indonesia yang sampelnya telah dipublikasikan di database genomik dipilih untuk penelitian. Data berikut akan menjalani beberapa protokol bioinformatika, divisualisasikan ke dalam pohon filogenetik, rendering 3D, dan penilaian dampak mutasi untuk dianalisis.HasilTerdapat 25 clade unik dan 318 RBD unik di Indonesia, mulai dari sampel paling awal hingga tahun 2022. T478K merupakan mutasi RBD yang paling sering, sedangkan 22B merupakan clade yang paling banyak diamati di Indonesia. Varian omicron menunjukkan skor docking yang lebih rendah dan destabilisasi protein yang lebih tinggi serta Kd yang lebih tinggi daripada galur tipe delta dan liar.KesimpulanHasil dari penelitian tersebut menunjukkan tren penurunan patogenisitas virus kemungkinan sebagai pertukaran untuk peningkatan penularan karena mutasi pada RBD selama bertahun-tahun.

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Introduction

Since the beginning of the outbreak, the dynamic mutations on the RBD domain in the SARS-CoV-2 spike protein have altered the pathogenicity of variants circulating in Indonesia. This research analyzed the mutation trend on the various sample RBD domains in Indonesia published in Genomic Databases using genomic profiling.

Method

Patients infected with COVID-19 in Indonesia with samples published in genomic databases are selected for the research. The following data underwent several bioinformatics protocols, visualized into phylogenetic trees, 3D rendering, and assessment of mutational impact for analysis.

Results

There are 25 unique clades and 318 unique RBD in Indonesia, ranging from the earliest sample to 2022. T478K was the most frequent RBD mutation, while 22B was the most abundant clade observed in Indonesia. The omicron variant showed a lower docking score, higher protein destabilization, and higher Kd than the delta and wild-type strains. Conclusion

The results from the study suggested a decreasing trend in the virus pathogenicity as a potential trade-off to increased transmissibility due to the mutations in RBD throughout the years."