1. The 6th International Congress of Clinical Laboratory and Clinic
Cloning and Expression of FHBP Protein of Neisseria meningitidis in E. coli
Yarian, F.*, Bandehpour, M.1, Kazemi, B.1 ,Ahangarzadeh, Sh.1
Shahid Beheshti University of Medical Sciences, Medical college, Biotechnology Department *
Background and Objective:
Meningitis is the inflammation or infection of the membranes that is covering the brain or spinal cord. Bacterial meningitis have seen in children older than five years and it is typically more dangerous than virus and it can cause secondary problems in many cases in brain and nervous system. If it is diagnosed and treated early, Approximately ninety percent of children recover. Most common cause of bacterial meningitis in children is" Neisseria meningitidis " . Now if you suspect meningitis, even before laboratory results become available starting treatment with antibiotics.
Fig1-Vector containing a FHBP
gene construct
Fig2-PQE 30 Vectore
Material and methods: M
Cut
Un cut
FHBP
In this study, A DNA encoding the FHBP of Neisseria meningitidis was amplified by PCR using specific primers with appropriate restriction enzyme sites in their ends. The gene was cloned in the pQE30 vector and transformed to E. coli strain M15 and expressed in optimal condition.
3000 bp
860 bp
1000 bp
860 bp
Findings:
470 bp
500 bp
The desired gene is 860 nucleotides and it was Successfully PCR and cloned in pQE30 vector with enzymatic digestion, and it was confirmed by sequencing with universal primers and sequences were obtained for final approval. Protein obtained were approved by SDS-PAGE and Western blotting. This protein can be valuable for diagnosis of Meningitis
Fig3-PCR:FHBP gene
Fig4-digestion of vectore with
Sal I & kpn I
Fig5-Cloning confirmed by
restriction enzyme digestion
BL21 t0 t3 t5 t3 t5 M
BL21
Conclusion:
70 kd
We well cloned and expressed FHBP protein of Neisseria meningitidis in E. coli. The expressed protein was analyzed on SDS-PAGE and confirmed by western blotting using specific antibody to His tag. This protein can be valuable for diagnosis of Meningitis.
40 kd
32 kd
Key words:
Neisseria meningitidis bacteria, FHBP protein, cloning
Fig6-SDS-page:expression of protein in BL21
(0.5 mM IPTG)
Fig7-Western blotting: Confirm the protein
Expression With anti his tag antibody
Acknowledgment:
I want to express my gratitude to Shahid Beheshti University of Medical science and center of cellular and molecular researches of Shahid Beheshti’s Medical Sciences for suporting this research and also wish to extend my thanks to Dr. M. Bandehpour, Dr. B.Kazemi, Dr .Z.Sharifnia, Dr. Sh. Ahangarzadeh, Miss. A. Koochaki and Miss. Z.Soleimanifar for their helpful comments.
References:
1- Epidemic meningitis, meningococcaemia, and Neisseria meningitides- David S Stephens, Brian Greenwood, Petter Brandtzaeg
2-TZ YL, Martin LE,.Environmental survival of Neisseria meningitidis,Epidemol infect Jan;142(1):187-90
3- Feibig T, Berti F Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: A new perspective for vaccine development.Glicobiology Feb;24(2):150-8
Poster code:PBN-014