2. INTRODUCTIO
N
~Pseudomonas aeruginosa: is a common Gram-negative,
rod-shaped bacterium. As an oportunistic human pathogen
has a remarkable capacity to cause disease in susceptible
hosts. It shows inherent and acquired resistance to many
antimicrobial agents.
~ PilQ 380-706 : its a protein member of the so called
“secretin” family required for configuration of the outer
membrane pore through wich the pilus is extruded. It
consists of five conserved domains (secretin _N,secretin,
STN, HofQ and AMIN). This region facilitates the passage of
folded proteins, filamentous phage particles, DNA, and other
macromolecules across the outher membrane.
3. INTRODUCTI
ON
~Relation between P.aeruginosa and PilQ 380-706 :
The PilQ380-706 protein plays an important role in the
biogenesis of type 4 pili and thus, the primary
establishment of P. aeruginosa.
Type 4 pili (T4P) is an important virulence factor of
Pseudomonas aeruginosa .T4P pass the outer membrane
through a large oligomeric channel made of a single PilQ
protein that is most highly conserved at their C-termini,
this segment is a potential candidate for a new vaccine.
4. OBJETIVE
➤ To develop a molecular characterization and
Functional Analysis of the PilQ380-706
producing it as a recombinant protein
5. MATERIALS AND
METHODS
~Bacterial strains: Escherichia coli (E. coli) strains Top10F
and BL21 (DE3) were used as preservation and expression
hosts.
P. aeruginosa laboratory strain PAO1 were performed
~Plasmids: The recombinant plasmid pET28a/pilQ1138-
2118
~Enzymes for DNA manipulations
~HRP-conjugated goat anti-rabbit IgG
~Medium: The strains were cultured in LB broth or on agar.
6. MATERIALS AND METHODS
Construction of the expression vector:
After transformation of the recombinant vector into
E. coli Top10F competent cells, transformants were
screened on LB plates supplemented with
kanamycin. The recombinant vector was extracted
from E. coli
E. coli
expression
vector pET28a
+
The
pilQ1138-
2118 gene
T7 promoter
kanamycin-
resistant
gene
Gene containing BamHI
and HindIII sites at the
5′ and 3′ ends
respectively.
C and N-terminal sixHis-
tagged sequences
Start codon ATG
7. MATERIALS AND METHODS
1. Confirmation of the recombinant vector
Was used for : show the presence of the
pilQ gene in the construct
Design specific
primers for
pilQ1138-2118
secuence
Made the
amplification from
the recombinant
vector (PCR)
PCR: it is a technique that consist of
obtaining a set of genetic elements
identical to its precursor. The objetive Is
the amplification of genes or DNA or
indirectly through RNA
Principles
Double stranded DNA denaturation
Specific hybridization with a primer of
single stands
Single strand replication by a DNA
polymerase
The vector was treated with
restriction endonucleases
BamHI and HindIII
8. MATERIALS AND METHODS
2. Expression and isolation of
inclusion bodies : to overexpress the
protein
3. Solubilization, refolding , and
purification of r-PilQ 380-706
4. Preparation and purification of anti r-
PilQ 380-369 IgG: to determine the
inmunogenic nature of purified PilQ
5. Sds-PAGE electrophoresis : technique
used to separate and characterize proteins
Principle: separation of molecules by charge
and molecular weight
Was used to: evaluate the expression of the
r-PilQ 380-706 protein.
9. MATERIALS AND
METHODS
6. Western blot:In this technique a mixture of proteins is
separated based on molecular weight, and thus by type, through
gel electrophoresis. These results are then transferred to a
membrane producing a band for each protein. The membrane is
then incubated with labels antibodies specific to the protein of
interest.
Principle : based on the antibody antigen reaction.
Was used to : confirm the functional activities of the produced
PilQ380-360 protein and to determine the specificity of the
antiserum raised against purified r-PilQ380706.
13. Authors What they said or did
Is related to the
article
Middelberg AP.
Tsumoto K, Ejima
D, Kumagai
high-throughput protein-refolding techniques have been developed for
renaturation of inclusion bodies . These include dilution, dialysis or
solid-phase separation.
Matthey B et al
pET-series vectors also contain a lacI gene that provides lac
repressor molecules to downregulate both the lacUV5-controlled
chromosomal T7 RNA polymerase and the T7lac promoter
Singh SM, Panda
AK
Motility has been exhibited to be an important virulence factor in
microbial pathogenesis
Koo et al
Showed that the absence of twitching motility of P. aeruginosa is
correlated with the lack of PilQ multimer
CUSSION
14. CONCLUSION
S
1. The present study described a modified method for expression ,
purification and refolding of r-PilQ380-706.
2. PilQ as recombinant protein was biologically active and
recommended to be used as vaccine or and adjuvant.
Personal
1.The study carried out is probably going to be effective because the
recombinant protein that was produced is highly conserved among the
different species of P. areuginosa.
2. Despite the fact that bacterial attachment is not the only virulence
factor of P.areuginosa , by altering this mechanism, it is possible to
reduce its pathogenicity.
