ABSTRACT
Haps protein plays central role in initial interaction of nontypeable Haemophilus influenzae [NTHi] with human respiratory epithelial cells. While other surface-exposed proteins of NTHi are highly variable, The HapS domain is highly conserved among H. influenzae strains. Recent studies demonstrated that HapS adhesive activity resides within the C-terminal 311 amino acids of the protein and also they showed that the C-terminal 311 amino acids of HapS [C-Haps] are capable of eliciting a protective immune response against NTHi colonization. The pET24a-chaps plasmid harboring c-haps sequence from NTHi PTCC1766 was constructed. The amino acid sequences of C-Haps of this study was aligned with C-Haps of three NTHi strains [N187, TN106, P860295] and antigenicity plot of studied rC-Haps was done bioinformatic software. The pET24a-chaps expression was conducted in E.coli BL21 [D3E] and its expression was confirmed by SDS-PAGE and Western blotting methods. The rC-Haps was purified via immobilized metal affinity chromatography. Amino acid sequence alignment of rCHaps sequence of current study and rC-Haps from the NTHi strains N187, TN106, P860295 showed more than%97 identity. Antigenicity plot identified 9 common highly antigenic domains that were located exactly in conserved regions among 4 different NTHi strains. Due to presence of highly conserved antigenic epitopes among C-Haps of NTHi PTCC1766 and other NTHi strains, rC-Haps of current study could be theoretically a vaccine candidate against NTHi strains of different geographical areas
ABSTRACT
Nontypeable Haemophilus influenzae [NTHi] is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae [H. influenzae] Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called "HapS" and a 45 kDa Cterminal translocator domain called "Hapbeta". Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain [CBD] which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24alpha-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography [IMAC] using Ni-NTA columns. The highest expression level of target protein was achieved when CBD expressing E. coli BL21 [DE3] cells were grown at 37°C in 2xTY medium with 1.0 mM IPTG at mid-log phase [OD[600 nm] equal to 0.6] for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than%97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process