Cloning, expression, and purification of recombinant flagellin (r-fliC)‎ of Salmonella enterica serovar Typhimurium DT 104 and its role in protective immunity in BALBc mice

Other Title(s)

استنسال و تعبير و تنقية للبروتين السوطي الهجين لبكتريا Salmonella enterica serovar Typhimurium DT 104 و دوره في الحصانة الوقائية في فئران BALBc

Dissertant

al-Husayni, Anwar Ali Abd Allah

Thesis advisor

al-Qazzaz, Abd al-Karim Abd al-Razzaq Abd al-Wahhab
Melconian, Alice Krikor

University

University of Baghdad

Faculty

College of Science

Department

Biotechnology Department

University Country

Iraq

Degree

Ph.D.

Degree Date

2012

English Abstract

The present study was designed to clone flagellin gene (fliC) of the recently emerged Salmonella enterica serovar Typhimurium DT104 strain into Escherichia coli SG13009 (pRP4).

The genomic DNA was extracted from the Salmonella strain and fliC gene was amplified by polymerase chain reaction (PCR) using specific primers designed to complete sequence of fliC gene located on chromosomal DNA of the S.

Typhimurium according to the database available on National Center of Biotechnology Information (NCBI).

The results of electrophoresis on agarose gel indicated that the molecular size of fliC gene fragment was approximately, 1485 base pair (bp).

The amplified gene fragment was cloned into pQE30 vector after the fliC gene and pQE30 vector were digested by BamHI and HindIII to facilitate ligation of the gene with the vector, and then transforming fliC-pQE30 into E.

coli.

The results showed the presence of transformed colonies.

PCR screening of transformed colonies(fliC) were achieved by its use as template with specific primers.

Electrophoresis on agarose gel revealed 13 transformants to have one band with molecular size of approximately, 1485 bp.

To ensure the success of the cloning, fliC-pQE30 vector were digested with the BamHI and HindIII to obtain the components of the clone separately.

The electrophoresis on agarose gel revealed the presence of two bands: one belongs to fliC and the other to the vector in comparison with the marker.

To ensure that the fliC gene has the desired orientation in the recombinant vector, sequencing of the recombinant vector was conducted.

The results of sequencing suggested that the fliC gene has been inserted in the right direction in 4 transformants (3, 4, 9, and 13) of the 13 tested E.

coli transformants (3, 4, 6, 8, 9, 11, 13, 18, 19, 21, 23, 25, and 30).

The optimal expression condition for the II cloned flagellin gene in the transformed E.

coli was determined by subjecting the growing bacteria to several induction conditions using several concentrations of IPTG (0.5, 0.7 and 1 mM) and different time courses of induction (1, 2, 3, 4, 5 and 6) hours at 37°C.

The results showed that the optimal production of recombinant fliC product was achieved after induction of the genetically engineered E.

coli SG 13009 with 0.5 mM IPTG for 3 hr at 37°C for transformant No.

13.

Immobilized metal ion affinity chromatography (IMAC) with nickel divalent ions (Ni-NTA chromatographic) was used to separate and purify the recombinant flagellin using batch purification of 6x His-tagged proteins from E.

coli under denaturing conditions.

The results of electrophoresis on polyacrylamide gel suggested effectiveness of the protocol in obtaining flagellin protein high purity.

The approximate molecular size of fliC product was about 56 KDa in the form of sharp protein band in comparison with the same band of cell fractions without induction with IPTG.

To assess the immunogenicity of the recombinant flagellin, a dose of 30 micrograms were mixed with an adjuvant and were inoculated into a group of BALB/c mice whereas a similar group of mice were injected with the same solution but without the flagellin protein, as a control.

Three injections were administered into each mouse and blood was collected before each injection time to determine the anti-flagellin serum titers.

It was concluded that the recombinant flagellin has a good immunogenic properties.

The relative protective role of the anti-flagellin antibodies was assessed by subjecting the immunized and the control mice to a significant challenge dose of the parent S.

Typhimurium.

A larger number of the immunized mice survived the challenge dose of S.

Typhimurium than the control mice, suggesting the protective value of the recombinant flagellin as an immunogene and hence the III recommendation for its further assessment as a possible vaccine against infection with S.

Typhimurium in humans and animals.

Main Subjects

Biology

Topics

• Genetics
• Laboratory animals

No. of Pages

152

Table of Contents

• APA
• MLA
• AMA

Language

English

Data Type

Arab Theses

Record ID

BIM-601278