Recovering Immunogenic <i>Orthohantavirus puumalaense</i> N Protein from Pellets of Recombinant <i>Escherichia coli</i>
(1) Background: Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in Eurasia. <i>Orthohantavirus puumalaense</i> (PUUV), carried by bank voles (<i>Myodes glareolus</i>), is the principal zoonotic pathogen of HFRS in this region. Despite ongoing efforts...
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MDPI AG
2025-07-01
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| author | Natalya Andreeva Ekaterina Martynova Polina Elboeva Milana Mansurova Ilnur Salafutdinov Aleksandr Aimaletdinov Rafil Khairullin Diksha Sharma Manoj Baranwal Sara Chandy Dilbar Dalimova Alisher Abdullaev Mirakbar Yakubov Albert Rizvanov Svetlana Khaiboullina Yuriy Davidyuk Emmanuel Kabwe |
| author_facet | Natalya Andreeva Ekaterina Martynova Polina Elboeva Milana Mansurova Ilnur Salafutdinov Aleksandr Aimaletdinov Rafil Khairullin Diksha Sharma Manoj Baranwal Sara Chandy Dilbar Dalimova Alisher Abdullaev Mirakbar Yakubov Albert Rizvanov Svetlana Khaiboullina Yuriy Davidyuk Emmanuel Kabwe |
| author_sort | Natalya Andreeva |
| collection | DOAJ |
| description | (1) Background: Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in Eurasia. <i>Orthohantavirus puumalaense</i> (PUUV), carried by bank voles (<i>Myodes glareolus</i>), is the principal zoonotic pathogen of HFRS in this region. Despite ongoing efforts to develop effective drugs and vaccines against PUUV, this challenge remains. (2) Aim: In this study, we aimed to express a large quantity of the PUUV recombinant N (rN) protein using <i>E. coli.</i> We also sought to develop a protocol for extracting the rN protein from pellets, solubilizing, and refolding it to restore its native form. This protocol is crucial for producing a large quantity of rN protein to develop vaccines and diagnostic tools for HFRS. (3) Methods; PUUV S segment open reading frame (ORF) coding for N protein was synthesized and cloned into the plasmid vector pET-28 (A+). The ORF was transformed, expressed and induced in BL21(DE3) pLysS <i>E. coli</i> strain. Subsequently, rN protein was purified using immobilized metal affinity and ion chromatography. Immune reactivity of rN protein was tested by employing in house and commercial VektoHanta-IgG kit ELISA methods (both in vitro and in vivo). (4) Results: The best conditions for scaling up the expression of the PUUV rN protein were an incubation temperature of 20 °C during a 20 h incubation period, followed by induction with 0.5 mM IPTG. The most significant protein yield was achieved when the pellets were incubated in denaturing buffer with 8M urea. The highest yield of refolded proteins was attained using non-denaturing buffer (50 mM Tris-HCl) supplemented with arginine. A final 50 μL of PUUV rN protein solution with a concentration of 7 mg/mL was recovered from 1 L of culture. The rN protein elicited an antibody response in vivo and reacted with serum taken from patients with HFRS by ELISA in vitro. (5) Conclusion: Therefore, the orthohantavirus N protein’s ability to elicit immune response in vivo suggests that it can be used to develop vaccines against PUUV after conducting in vitro and in vivo studies to ascertain neutralising antibodies. |
| format | Article |
| id | doaj-art-8e73f8c7460348598da1cf3430b671f7 |
| institution | DOAJ |
| issn | 2076-393X |
| language | English |
| publishDate | 2025-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj-art-8e73f8c7460348598da1cf3430b671f72025-08-20T02:47:06ZengMDPI AGVaccines2076-393X2025-07-0113774410.3390/vaccines13070744Recovering Immunogenic <i>Orthohantavirus puumalaense</i> N Protein from Pellets of Recombinant <i>Escherichia coli</i>Natalya Andreeva0Ekaterina Martynova1Polina Elboeva2Milana Mansurova3Ilnur Salafutdinov4Aleksandr Aimaletdinov5Rafil Khairullin6Diksha Sharma7Manoj Baranwal8Sara Chandy9Dilbar Dalimova10Alisher Abdullaev11Mirakbar Yakubov12Albert Rizvanov13Svetlana Khaiboullina14Yuriy Davidyuk15Emmanuel Kabwe16Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaDepartment of Biotechnology, Thapar Institute of Engineering and Technology, Patiala 147001, IndiaDepartment of Biotechnology, Thapar Institute of Engineering and Technology, Patiala 147001, IndiaThe Childs’ Trust Medical Research Foundation (CTMRF) Kanchi Kamakoti Childs Trust Hospital (KKCTH), Chennai 600034, IndiaCenter for Advanced Technologies, Tashkent 100174, UzbekistanCenter for Advanced Technologies, Tashkent 100174, UzbekistanCenter for Advanced Technologies, Tashkent 100174, UzbekistanInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, RussiaInstitute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, Russia(1) Background: Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in Eurasia. <i>Orthohantavirus puumalaense</i> (PUUV), carried by bank voles (<i>Myodes glareolus</i>), is the principal zoonotic pathogen of HFRS in this region. Despite ongoing efforts to develop effective drugs and vaccines against PUUV, this challenge remains. (2) Aim: In this study, we aimed to express a large quantity of the PUUV recombinant N (rN) protein using <i>E. coli.</i> We also sought to develop a protocol for extracting the rN protein from pellets, solubilizing, and refolding it to restore its native form. This protocol is crucial for producing a large quantity of rN protein to develop vaccines and diagnostic tools for HFRS. (3) Methods; PUUV S segment open reading frame (ORF) coding for N protein was synthesized and cloned into the plasmid vector pET-28 (A+). The ORF was transformed, expressed and induced in BL21(DE3) pLysS <i>E. coli</i> strain. Subsequently, rN protein was purified using immobilized metal affinity and ion chromatography. Immune reactivity of rN protein was tested by employing in house and commercial VektoHanta-IgG kit ELISA methods (both in vitro and in vivo). (4) Results: The best conditions for scaling up the expression of the PUUV rN protein were an incubation temperature of 20 °C during a 20 h incubation period, followed by induction with 0.5 mM IPTG. The most significant protein yield was achieved when the pellets were incubated in denaturing buffer with 8M urea. The highest yield of refolded proteins was attained using non-denaturing buffer (50 mM Tris-HCl) supplemented with arginine. A final 50 μL of PUUV rN protein solution with a concentration of 7 mg/mL was recovered from 1 L of culture. The rN protein elicited an antibody response in vivo and reacted with serum taken from patients with HFRS by ELISA in vitro. (5) Conclusion: Therefore, the orthohantavirus N protein’s ability to elicit immune response in vivo suggests that it can be used to develop vaccines against PUUV after conducting in vitro and in vivo studies to ascertain neutralising antibodies.https://www.mdpi.com/2076-393X/13/7/744N proteinhemorrhagic fever with renal syndrome<i>Orthohantavirus puumalaense</i>vaccineimmunogenicityimmunization |
| spellingShingle | Natalya Andreeva Ekaterina Martynova Polina Elboeva Milana Mansurova Ilnur Salafutdinov Aleksandr Aimaletdinov Rafil Khairullin Diksha Sharma Manoj Baranwal Sara Chandy Dilbar Dalimova Alisher Abdullaev Mirakbar Yakubov Albert Rizvanov Svetlana Khaiboullina Yuriy Davidyuk Emmanuel Kabwe Recovering Immunogenic <i>Orthohantavirus puumalaense</i> N Protein from Pellets of Recombinant <i>Escherichia coli</i> Vaccines N protein hemorrhagic fever with renal syndrome <i>Orthohantavirus puumalaense</i> vaccine immunogenicity immunization |
| title | Recovering Immunogenic <i>Orthohantavirus puumalaense</i> N Protein from Pellets of Recombinant <i>Escherichia coli</i> |
| title_full | Recovering Immunogenic <i>Orthohantavirus puumalaense</i> N Protein from Pellets of Recombinant <i>Escherichia coli</i> |
| title_fullStr | Recovering Immunogenic <i>Orthohantavirus puumalaense</i> N Protein from Pellets of Recombinant <i>Escherichia coli</i> |
| title_full_unstemmed | Recovering Immunogenic <i>Orthohantavirus puumalaense</i> N Protein from Pellets of Recombinant <i>Escherichia coli</i> |
| title_short | Recovering Immunogenic <i>Orthohantavirus puumalaense</i> N Protein from Pellets of Recombinant <i>Escherichia coli</i> |
| title_sort | recovering immunogenic i orthohantavirus puumalaense i n protein from pellets of recombinant i escherichia coli i |
| topic | N protein hemorrhagic fever with renal syndrome <i>Orthohantavirus puumalaense</i> vaccine immunogenicity immunization |
| url | https://www.mdpi.com/2076-393X/13/7/744 |
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