Brucella melitensis rev.1 ΔOmp 19 marker aşı geliştirilmesi
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Dosyalar
Tarih
2020
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Brusella, birçok konak üzerinde etkili hücre içi zoonoz bir patojendir. Canlı attenüe suş olan B. melitensis Rev.1aşı suşu, piyasada koyun-keçi brusellozisine karşı en etkin aşı suşudur. Koruma gücü açısından koyun-keçilerde hatta sığırlarda güvenle kullanılabilmektedir. Ancak koyun-keçilerde saha enfeksiyonlarından/aşılamaya bağlı oluşan antikorları ayırt edebilmesine yönelik etkin bir aşı suşu olmamakla ile birlikte birçok çalışma mevcuttur. Brusella dış membranında bulunan Omp19 proteini hem virulans hem de koruma ile ilişkili bir antijendir. Bu çalışmada 534 bp uzunluğunda Omp19 ORF silinmesi için 5’ve 3’ homoloji bölgesini bulunduran pJQ200KS isimli intihar plazmidi elektroporasyon ile penisilin, ampisilin ve glisin ile sferoplast haline getirilen B. melitensis Rev.1’lere aktarımı sağladı. Delesyonu doğrulanan mutant suştan (B. melitensis Rev.1 ΔOmp19) aşı hazırlandı. Fare modellerinde B. melitensis Rev.1 aşısı ile etkinlik, immün temizlenme ve Omp19 bölge delesyonunun yarattığı enfeksiyonlardan ayrımının kıyaslanması amaçlandı. Gen aktarım sürecinde E.coli’de mutant vektör oluşturmak için ışı şok transformasyon metodu X-gal’lı besi yerinde mavi beyaz koloni ayrımı, Brusella’ya gen aktarımı için elektroporasyon kullanıldı. Sferoplast dönüşümünü gözlemlemek için S.E.M. elektron mikroskobundan yararlanıldı. Mutant vektör ve delesyon mutantlar PZR ve dizi analizleri ile doğrulandı. Fare model denemeleri bireysel havalandırmalı kafes sistemi içerisinden yürütüldü. Elektroporasyon sonucunda 17, 43, 44, 45 (glisin sferoplast) ve 19, 46 (ampisilin sferoplast) isimli denemelerde gentamisine dirençli B. melitensis Rev.1’ler tespit edildi. Fare modeli etkinlik denemelerinde, 3 ticari aşı OIE standartlarına uygun uyarım sağladığı tespit edildi. Delesyon mutantlarından 19 ve 44-10’nun uyarım gücü açısında ticari aşılarla benzer etkiliğe sahip olmasına karşın Omp19 proteini ile yapılan ELISA testinde negatif kontrolden farksız sonuç tespit edildi. Çalışmada elde edilen 2 adet Rev.1 Omp 19 delesyon mutantı fare modelinde en az ticari aşılar kadar yeterli rezidüel virülans ve protektif immunite oluşmaktadır. Omp19 geni silinmiş B. melitensis Rev.1 ΔOmp19 suşundan aşı hazırlanılması ve kullanılması durumunda serolojik olarak Omp19 proteini taranmasına yönelik ELISA testleri ile aşılı/enfekte ayrımı (DIVA) yapılabileceği tespit edilmiştir.
Brucella is an intracellular zoonotic pathogen that acts on many hosts. B. melitensis Rev.1, a live attenuated strain, is the most effective vaccine strain against sheep-goat brucellosis in the market. It can be used safely in sheep-goats and even cattle against antibody titer and challenge. However, there is not an effective vaccine strain in sheep-goats to distinguish antibody titer from field infections/vaccination, but there are many studies. The Omp19 region on the brucella outer membrane is both virulence and a protective antigen. In this study, the suicide plasmid pJQ200KS, which contains 5 and 3 'homology region for the removal of 534 bp Omp19 ORF, was transferred to B. melitensis Rev.1, which was transformed into spheroplasts with penicillin, ampicillin, and glycine in electroporation. Vaccines verified deletion mutant mouse models in fields inTurkey B. melitensis Rev. 1 vaccines with activity aimed to infection by comparison of the distinction of the immune clearance of deletions and Omp19.In the gene, the transfer process, the blue and white colony separation was used in breeding the site with X-gal in the light shock transformation method to create a mutant a vector in E.coli, and electroporation was used for gene transfer to Brucella.S.E.M. to observe the spheroplast transformation. the electron microscope was used. Mutant vector and deletion mutants were confirmed by PCR and sequence analysis. Mouse model trials were conducted through the IVS cage system. As a result of electroporation, gentamicin resistant B. melitensis Rev.1 was detected in trials named 17, 43, 44, 45 (glycine spheroplast) and 19, 46 (ampicillin spheroplast). In mouse model efficacy trials, 3 commercial vaccines were found to comply with OIE standards. Although the deletion mutants had similar efficacy with commercial vaccines in terms of stimulation power of 19 and44-10, the ELISA test with Omp19 protein showed no different results from the negative control. The 2 Rev.1 Omp19 deletion mutants obtained in the study consisted of sufficient residual virulence and protective immunity as at least as commercial vaccines. With this study, it has been determined that if the vaccine is prepared and used from B.melitensis Rev.1 strain whose ΔOmp19 gene has been deleted, the vaccine / infected separation (DIVA) can be performed by ELISA tests for screening Omp19 protein.
Brucella is an intracellular zoonotic pathogen that acts on many hosts. B. melitensis Rev.1, a live attenuated strain, is the most effective vaccine strain against sheep-goat brucellosis in the market. It can be used safely in sheep-goats and even cattle against antibody titer and challenge. However, there is not an effective vaccine strain in sheep-goats to distinguish antibody titer from field infections/vaccination, but there are many studies. The Omp19 region on the brucella outer membrane is both virulence and a protective antigen. In this study, the suicide plasmid pJQ200KS, which contains 5 and 3 'homology region for the removal of 534 bp Omp19 ORF, was transferred to B. melitensis Rev.1, which was transformed into spheroplasts with penicillin, ampicillin, and glycine in electroporation. Vaccines verified deletion mutant mouse models in fields inTurkey B. melitensis Rev. 1 vaccines with activity aimed to infection by comparison of the distinction of the immune clearance of deletions and Omp19.In the gene, the transfer process, the blue and white colony separation was used in breeding the site with X-gal in the light shock transformation method to create a mutant a vector in E.coli, and electroporation was used for gene transfer to Brucella.S.E.M. to observe the spheroplast transformation. the electron microscope was used. Mutant vector and deletion mutants were confirmed by PCR and sequence analysis. Mouse model trials were conducted through the IVS cage system. As a result of electroporation, gentamicin resistant B. melitensis Rev.1 was detected in trials named 17, 43, 44, 45 (glycine spheroplast) and 19, 46 (ampicillin spheroplast). In mouse model efficacy trials, 3 commercial vaccines were found to comply with OIE standards. Although the deletion mutants had similar efficacy with commercial vaccines in terms of stimulation power of 19 and44-10, the ELISA test with Omp19 protein showed no different results from the negative control. The 2 Rev.1 Omp19 deletion mutants obtained in the study consisted of sufficient residual virulence and protective immunity as at least as commercial vaccines. With this study, it has been determined that if the vaccine is prepared and used from B.melitensis Rev.1 strain whose ΔOmp19 gene has been deleted, the vaccine / infected separation (DIVA) can be performed by ELISA tests for screening Omp19 protein.
Açıklama
Anahtar Kelimeler
Brucella melitensis, İntihar Plazmid, Suicide Plasmid
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Uslu, A. (2020). Brucella Melitensis Rev.1 ΔOmp 19 Marker Aşı Geliştirilmesi. (Doktora Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
