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

Year 2020, , 1 - 9, 17.03.2020

Burcu Emine Tefon Öztürk

https://doi.org/10.35414/akufemubid.576171

Abstract

Boğmaca,
gram-negatif Bordetella pertussis bakterisinin etken olduğu, akut,
oldukça bulaşıcı bir solunum yolu hastalığıdır. Şiddetli, kasılmalı ve uzun
süreli öksürük boğmacanın en tipik belirtisidir. En çok yeni doğanlarda görülen
bu hastalığın bu yaş grubunda ölümle sonuçlanabildiği de bilinmektedir. Bu
çalışmada, ileride aşı geliştirme araştırmalarında kullanılmak üzere, daha önce
immünojenik olduğu bilinen Lösin/ İzolösin/ Valin bağlanma proteini, Tip 2
fimbriya (Fim2) ve B. pertussis’in patojenitesinde önemli 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 gen bölgeleri ilk olarak bir klonlama vektörü
olan pGEM®-T Easy vektörüne klonlanmış ve E. coli DH5α hücrelerine
transforme edilmiş, ardından pET-28a(+) ekspresyon vektörüne atılarak E.
coli BL21(DE3)’de ekspresyonları yapılmıştır. Ekspresyonları yapılan
proteinler His-tag kolonları kullanılarak saflaştırılmıştır.

Keywords

Bordetella pertussis, BopD, BopN, LivJ, Fim2

Supporting Institution

TÜBİTAK, Akdeniz Üniversitesi

Project Number

115Z683, FBA-2015-644

Thanks

Bu projenin bir kısmı TÜBİTAK tarafından 115Z683 proje numarasıyla, bir kısmı Akdeniz Üniversitesi tarafından FBA-2015-644 proje numarasıyla desteklenmiştir.

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)

Abstract

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.

Keywords

Bordetella pertussis, BopD, BopN, LivJ, Fim2

Project Number

115Z683, FBA-2015-644

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