Bordetella pertussis’e Ait Varsayımsal Dış Zar Proteini D (BopD), Varsayımsal Dış Zar Proteini N (BopN), Lösin/İzolösin/Valin Bağlanma Proteini (LivJ) ve Tip 2 Fimbriya (Fim2)’nin Rekombinant Olarak İfade Edilmesi

Boğmaca, gram-negatif Bordetella pertussis bakterisinin etken olduğu, akut, oldukça bulaşıcı birsolunum yolu hastalığıdır. Şiddetli, kasılmalı ve uzun süreli öksürük boğmacanın en tipik belirtisidir. Ençok yeni doğanlarda görülen bu hastalığın bu yaş grubunda ölümle sonuçlanabildiği de bilinmektedir.Bu çalışmada, ileride aşı geliştirme araştırmalarında kullanılmak üzere, daha önce immünojenik olduğubilinen Lösin/İzolösin/Valin bağlanma proteini, Tip 2 fimbriya (Fim2) ve B. pertussis’in patojenitesindeönemli yer tutan Tip3 salgılama sisteminin (TTSS) elemanları olan varsayımsal dış zar proteini D (BopD)ve varsayımsal dış zar proteini N (BopN) rekombinant olarak eksprese edilmiştir. Proteinlere ait olan genbölgeleri ilk olarak bir klonlama vektörü olan pGEM®-T Easy vektörüne klonlanmış ve E. coli DH5αhücrelerine transforme edilmiş, ardından pET-28a(+) ekspresyon vektörüne atılarak E. coli BL21(DE3)’deekspresyonları yapılmıştır. Ekspresyonları yapılan proteinler His-tag kolonları kullanılaraksaflaştırılmıştır. SDS poliakrilamid jellerde BopD ve Fim2 proteinlerinin beklenenden daha yüksekmolekül ağırlığında bant verdiği görülmüştür. BopN ve BopD’nin ise indüklenmemiş örneklerde de kaçakprotein olarak ifade edildiği belirlenmiştir. Patojenitede önemli yer tutan bu proteinlerin rekombinantolarak ekspresyonları koruyuculuklarının belirlenebilmesi, söz konusu proteinleri içeren aşıkombinasyonlarının denenmesi ve yeni füzyon proteinlerinin oluşturulması gibi patojene karşı aşıgeliştirme çalışmalarında kullanılabilmelerini sağlaması açısından oldukça önem taşımaktadır.

Recombinant Expression of Bordetella pertussis Putative Outer Protein D (BopD), Putative Outer Protein N (BopN), Leucin/Isoleucine/Valin Binding Protein (LivJ) and Type 2 Fimbria (Fim2)

Whooping cough (pertussis) is a contagious upper respiratory tract infection primary causative agent of which is gram-negative coccobacillus Bordetella pertussis. This disease, which may be fatal especially in infants, is characterized by repeated, violent coughing spasms caused by infection and may results in death. In this study, selected immunogenic proteins, namely Leucin/Isoleucin/Valin binding protein (LivJ), Fimbria type 2 (Fim2) and members of Type 3 Secretion System (TTSS) Putative Outer Protein D (BopD) and Putative Outer Protein N (BopN) expressed recombinantly. Genes of selected proteins were first cloned into pGEM®-T Easy vector and transformed to into E. coli DH5α cells, then cloned into the expression vector pET-28a(+) and transformed into E. coli BL21(DE3) cells to express the proteins. Expressed proteins were also purified by using His-tag columns. The molecular weights of the expressed BopD and Fim2 proteins were higher than expected on SDS-PAGE. Leakage was observed during BopD and BopN protein expression. Recombinant expression of these proteins, which take place in pathogenicity, is really important because of their potential as a vaccine candidate. After detection the protective capacity of these proteins, it might be possible to use them in combinations or as fusions in vaccine development studies.

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Alger H.M. and Williams D.L., 2002. The disulfide redox system of Schistosoma mansoni and the importance of a multifunctional enzyme, thioredoxin glutathione reductase. Molecular and Biochemical Parasitology, 121(1), 129-139.
Benz I. and Schmidt A., 2002. Never say never again: protein glycosylation in pathogenic bacteria. Molecular Microbiology, 45(2), 267-276.
Berbers G.A., de Greeff S.C., Mooi F.R., 2009. Improving pertussis vaccination 2009. Human Vaccines, 5, 497–503.
Briand L., Marcion G., Kriznik A., Heydel J.M., Artur Y., Garrido C.,Seigneuric R., Neiers F., 2016. A self-inducible heterologous protein expression system in Escherichia coli. Scientific Reports, 6, 33037.
Cherry J.D. and Heininger U., 2004. Pertussis and other Bordetella infections in Textbook of Pediatric Infectious Diseases¸ edited by R. D. Feigin, J.D. Cherry, G.J. Demmler, and S. Kaplan, 5th ed. (The W. B. Saunders Co), 1588–1608.
Crowcroft N.S. and Britto J., 2002. Whooping cough-a continuing problem. British Medical Journal, 4, 1537–1538.
de Gouw D., Diavatopoulos D.A., Bootsma H.J., Hermans P.W.M., Mooi F.R., 2011. Pertussis: a matter of immune modulation. Federation of Europena Microbological Society Microbiology Reviews, 35(3), 441–474.
Gopal G.J. and Kumar A., 2013. Strategies for the production of recombinant protein in Escherichia coli. The Protein Journal, 32(6),419-425.
Kumar T.K.S., Gopalakrishna K., Prasad V.V.H., Pandit M.W., 1993. Multiple bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of proteins due to intermolecular disulfide cross-linking. Analytical Biochemistry, 213(2), 226-228.
Laemmli UK., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259),680-685.
Li H., Chengwei H., Daqing X., 2017. Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli. Electronic Journal of Biotechnology, 30, 88-94.
Mooi F. R., 2009. Bordetella pertussis strains with increased toxin production associated with pertussis resurgence. Emerging Infectious Diseases, 15(8), 1206–1213.
Mudd E.A., Sullivan S., Gisby M.F., Mironov A., Kwon C.S., Chung W.I., Day A., 2008. A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis. Journal of Experimental Botany, 59(10), 2597-2610.
Nagamatsu K., Kuwae A., Konaka T., Nagai S., Yoshida S., Eguchi M., Watanabe M., Mimuro H., Koyasu S., Abe A., 2009. Bordetella evades the host immune system by inducing IL‐10 through a type III effector, BopN. Journal of Experimental Medicine, 206, 3073–3088.
Rainbow L., Platt G.M., Simpson G.R., Sarid R., Gao S.J., Stoiber H. et al., 1997. The 222- to 234-kilodalton Latent Nuclear Protein (LNA) of Kaposi’s Sarcoma-Associated Herpesvirus (HumanHerpesvirus 8) is encoded by orf73 and is a component of the Latency-Associated Nuclear Antigen. Journal of Virology, 71(8), 5915-5921.
Ramagli L.S. and Rodrigez L.V., 1985. Quantitation of microgram amounts of protein in two-dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis, 6, 559–563.
Rath A., Glibowicka M., Nadeau V.G., Chen G., Deber C.M., 2008. Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proceedings of the National Academy of Sciences, 106(6), 1760-1765.
Sato Y. and Arai H., 1972. Leucocytosis promoting factor of Bordetella pertussis. Infection and Immunity, 6, 899–904.
Storm M., Advani A., Pettersson M., Hallander H.O., Bondeson K., 2006. Comparison of real time PCR and pyrosequencing for typing Bordetella pertussis toxin subunit 1 variants. Journal of Microbiological Methods, 65, 153-158.
Tefon B.E., Maass S., Ozcengiz E., Becher D., Hecker M. and Ozcengiz G., 2011. A comprehensive analysis of Bordetella pertussis surface proteome and identification of new immunogenic proteins. Vaccine, 29(19), 3583–3595.
Tefon B.E.,Ozcengiz E., Ozcengiz G., 2013. Pertussis vaccines: State of the art and future trends. Current Topics in Medicinal Chemistry, 13(20), 2581-2596. Yu X.J., Crocquet-Valdes P., Walker D.H., 1997. Cloning and sequencing of the gene for a 120-kDa imunoproteomic protein of Ehrlichia chaffeensis. Gene, 184, 2149-2154.
Zhu Y.Z., Cai C.S., Zhang W., Guo H.X., Zhang J.P., Ji Y.Y. et al., 2010. Immunoproteomic analysis of human serological antibody responses to vaccination with whole-cell pertussis vaccine (WCV).PLoS One, 5(11), e13915.
http://www.who.int/immunization/topics/pertussis/en/, (16.05.2018)