Öz
Bruselloz hayvanlardan veya hayvansal ürünlerden insanlara bulaşan en
önemli zoonotik hastalıklardan birisidir. Brucella
melitensis, brusella cinsi içerisinde en patojen türlerden birisidir. B. melitensis'in dış membran proteini 31
(Omp31) koruyucu bir immünojen ve önemli bir aday aşı olarak kabul
edilmektedir. Bu çalışmada, B. melitensis
Rev 1 suşundan Omp31’i kodlayan genin (omp31)
klonlanması amaçlandı. Çalışmada materyal olarak B. melitensis REV-1 canlı aşı suşu kullanıldı. Suşun
canlandırılmasının ardından, DNA ekstraksiyonu yapıldı. Dış membran proteini
kodlayan gen bölgesi, gen bankasından bulundu. Bu bölgeyi klonlamak için
gerekli primerler manuel olarak dizayn edildi. Primer dizaynından sonra
polimeraz zincir reaksiyonu yoluyla çoğaltılan gen bölgesi, pET28a ekspresyon vektörüne
klonlanarak Escherichia coli BL21
bakterisine transformasyonu gerçekleştirildi. E. coli BL21’den plazmid ekstraksiyonu yapıldı. Klonlama için
kullanılan restriksiyon enzimi ile kesim sonucunda insert ve plazmid ayrı
olarak gözlemlendi. Beklenen büyüklükte (790 bp) bulunan insert, tekrar
klonlama primeri ile çoğaltılarak doğrulamak amacıyla sekans analizi yapıldı.
Sekans analizi sonrasında elde edilen dizi gen bankası ile karşılaştırıldı ve
insertin B. melitensis dış membran
proteini olduğu doğrulandı. Bundan sonraki yapılacak çalışmalarla da klonlanan
rekombinant dış membran proteini Omp31 (rOmp31)’in antijenik özelliğinin
araştırılarak aşı adayı olma potansiyelinin belirlenmesi gerekmektedir.
Anahtar Kelimeler
Kaynakça
- Cassataro J, Pasquevich KA, Estein SM, et al. (2007): A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev. 1 vaccination. Vaccine, 25, 4437-46.
- Cloeckaert A, Jacques I, Grilló MJ, et al. (2004): Development and evaluation as vaccines in mice of Brucella melitensis Rev.1 single and double deletion mutants of the bp26 and omp31 genes coding for antigens of diagnostic significance in ovine brucellosis. Vaccine, 29, 2827-35.
- Cloeckaert A, Vizcaino N, Paquet JY, et al. (2002): Major outer membrane proteins of Brucella spp.: past, present and future. Vet Microbiol, 90, 229-47.
- Ding XZ, Bhattacharjee A, Nikolich MP, et al. (2005): Cloning, expression, and purification of Brucella suis outer membrane proteins. Protein Expr Purif, 40, 134-41.
- Dubray G, Bezard G (1980): Isolation of three Brucella abortus cell-wall antigens protective in murine experimental brucellosis. Ann Vet Res, 11, 367- 73.
- Gıda, Tarım ve Hayvancılık Bakanlığı Gıda ve Kontrol Genel Müdürlüğü (2012): Brusellanın konjuktival aşı ile kontrol ve eradikasyonu projesi. Genelge No: 2012/03.
- Kittelberger R, Hilbink F, Hansen MF, et al. (1995): Identification and characterization of immunodominant antigens during the course of infection with Brucella ovis. J Vet Diagn Invest, 7, 210-8.
- Monreal D, Grilló MJ, González D, et al. (2003): Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model. Infect Immun, 71, 3261-71.
- Office International des Epizooties (OIE) (2016): World Organization for Animal Health: Chapter 2.1.4. Brucellosis. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.04_BRUCELLOSIS.pdf.
- Sambrook J, Russell D (2001): Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
- Shojaei M, Tahmoorespur M, Soltani M, et al. (2018): In silico cloning and bioinformatics study of Brucella melitensis Omp31 antigen in different mammalian expression vectors. J Livest Sci Technol, 6, 65-76.
- Turner PC, Mclennan AG, Bates AD, et al. (2004): Moleküler Biyoloji Önemli Notlar. Nobel Yayınları.
- Vahedi F, Talebi AF, Ghorbani E, et al. (2011): Isolation, cloning and expression of the Brucella melitensis Omp31 gene. Iran J Vet Res, 12, 156-62.
Öz
Brucellosis is one of the most important zoonotic diseases transmitted
to humans from animals or animal products. Brucella
melitensis is the most pathogenic species in the genus brucella. The outer
membrane protein 31 (Omp31) of B.
melitensis is considered to be a protective immunogen and an important
candidate vaccine. In this study, cloning
from B. melitensis Rev 1 strain of Omp31
coding gene (omp31) was aimed. Brucella melitensis REV-1 live vaccine strain was used in the
study. After reactivation of strain, DNA was extracted from bacterial culture.
Gene sequence which encodes outer membrane protein was obtained from Gene Bank.
Primers were designed to clone this region. After primer design, the gene
region amplified by the polymerase chain reaction was cloned into the pET28a
expression vector and transformed into Escherichia
coli BL21 bacteria. Plasmid extraction was performed from E. coli BL21. The insert and plasmid were
separately observed as a result of cutting with the restriction enzyme used for
cloning. Expected size (790 bp) insert amplified with cloning primers again and
amplicon was sequenced. The sequence obtained after sequencing analysis was
compared to the gene bank and confirmed to be the outer membrane protein of B. melitensis. Further studies are
required to investigate the antigenic properties of the cloned recombinant
outer membrane protein Omp31 (rOmp31) and determine the potential to be a
candidate for vaccination.
Anahtar Kelimeler
Kaynakça
- Cassataro J, Pasquevich KA, Estein SM, et al. (2007): A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev. 1 vaccination. Vaccine, 25, 4437-46.
- Cloeckaert A, Jacques I, Grilló MJ, et al. (2004): Development and evaluation as vaccines in mice of Brucella melitensis Rev.1 single and double deletion mutants of the bp26 and omp31 genes coding for antigens of diagnostic significance in ovine brucellosis. Vaccine, 29, 2827-35.
- Cloeckaert A, Vizcaino N, Paquet JY, et al. (2002): Major outer membrane proteins of Brucella spp.: past, present and future. Vet Microbiol, 90, 229-47.
- Ding XZ, Bhattacharjee A, Nikolich MP, et al. (2005): Cloning, expression, and purification of Brucella suis outer membrane proteins. Protein Expr Purif, 40, 134-41.
- Dubray G, Bezard G (1980): Isolation of three Brucella abortus cell-wall antigens protective in murine experimental brucellosis. Ann Vet Res, 11, 367- 73.
- Gıda, Tarım ve Hayvancılık Bakanlığı Gıda ve Kontrol Genel Müdürlüğü (2012): Brusellanın konjuktival aşı ile kontrol ve eradikasyonu projesi. Genelge No: 2012/03.
- Kittelberger R, Hilbink F, Hansen MF, et al. (1995): Identification and characterization of immunodominant antigens during the course of infection with Brucella ovis. J Vet Diagn Invest, 7, 210-8.
- Monreal D, Grilló MJ, González D, et al. (2003): Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model. Infect Immun, 71, 3261-71.
- Office International des Epizooties (OIE) (2016): World Organization for Animal Health: Chapter 2.1.4. Brucellosis. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.04_BRUCELLOSIS.pdf.
- Sambrook J, Russell D (2001): Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
- Shojaei M, Tahmoorespur M, Soltani M, et al. (2018): In silico cloning and bioinformatics study of Brucella melitensis Omp31 antigen in different mammalian expression vectors. J Livest Sci Technol, 6, 65-76.
- Turner PC, Mclennan AG, Bates AD, et al. (2004): Moleküler Biyoloji Önemli Notlar. Nobel Yayınları.
- Vahedi F, Talebi AF, Ghorbani E, et al. (2011): Isolation, cloning and expression of the Brucella melitensis Omp31 gene. Iran J Vet Res, 12, 156-62.
Ayrıntılar
| Birincil Dil | Türkçe |
|---|---|
| Bölüm | Makaleler |
| Yazarlar | |
| Yayımlanma Tarihi | 26 Temmuz 2019 |
| Gönderilme Tarihi | 9 Nisan 2019 |
| Kabul Tarihi | 22 Temmuz 2019 |
| Yayımlandığı Sayı | Yıl 2019 Cilt: 30 Sayı: 2 |
Kaynak Göster
Kabul edilen makaleler Creative Commons Atıf-Ticari Olmayan Lisansla Paylaş 4.0 uluslararası lisansı ile lisanslanmıştır.