Cloning of conserved regions of nontypeable Haemophilus influenzae hmw1 core binding domain

Document Type : Research Article

Authors

• Sepideh Meidaninikjeh ¹
• Farzam Vaziri ²
• Seyed Davar Siadat ³

¹ Department of Microbiology, Faculty of Advanced Sciences & Technology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran - Iran (IAUPS)

² Assistant professor of Pasteur institute of Ira,Research Center of Microbiology, Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran.

³ Associated professor of Pasteur institute of Iran, Research Center of Microbiology, Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran.

Abstract

Colonization of nontypeable Haemophilus influenzae (NTHi) in nasopharynx causes respiratory tract disease. In 80% of clinical isolates, HMW proteins are the major adhesions and induce protective antibodies in the hosts. Therefore, it can be used as a vaccine candidate. The aim of this study is designing and cloning of the conserved regions of NTHi hmw1 core binding domain.In this study, the standard strain of NTHi (PTCC1766) was used and cultured on chocolate agar. Genomic DNA was extracted by commercial kit. Then, specific primers were designed for highly conserved regions and PCR product was purified. PCR product was inserted into the PTG19-T plasmid and the recombinant plasmids were transferred to Escherichia.coli (Top10). For screening the recombinant cells from others, they were cultured on LB agar which had ampicillin and tetracycline antibiotics. Recombinant cells were purified by a specific kit. The presence of conserved regions of NTHi hmw1 core binding domain was confirmed by colony-PCR, double digest and sequencing.
In this study, having analyzed the result of sequencing by Mega6 software, we understood that the sequencing result was same as our target area (Conserved regions of hmw1 core binding domain), indicating that the cloning process have been performed successfully. It is concluded that, due to the presence of high similarity among some regions of hmw1 core binding domain among NTHi PTCC1766 and other NTHi strains, core binding domain can be an appropriate candidate for subunit vaccines in case of inducing immune system response.

Keywords

• Nontypeable Haemophilus influenzae
• core binding domain of HMW1
• cloning
• recombinant plasmid
• colony-PCR
• double digest
• sequencing

Main Subjects

• Molecular Microbiology

---