Cloning of heat shock protein 60 gene (groEL)‎ of Salmonella Typhimurium and study its protective effect against the infection in BALBc mice

العناوين الأخرى

استنسال جين بروتين الصدمة الحرارية 60 (groEL)‎ لبكتريا Salmonella Typhimurium و دراسة تأثيره الوقائي ضد الخمج بها في فئران BALBc

مقدم أطروحة جامعية

Sad, Isra Farid

مشرف أطروحة جامعية

Aziz, Ghazi Munim
Adhiah, Ali Husayn

الجامعة

جامعة بغداد

الكلية

كلية العلوم

القسم الأكاديمي

قسم التقانة الإحيائية

دولة الجامعة

العراق

الدرجة العلمية

دكتوراه

تاريخ الدرجة العلمية

2012

الملخص الإنجليزي

The study was designed to clone the heat shock protein 60 (HSP60) groEL gene from two strains of Salmonella enterica serovar Typhimurium (LT2) ATCC 19585 and (DT104) 1166 in E.

coli by extracting the genomic DNA of these strains and the full-length coding region of groEL gene of Salmonella was amplified by polymerase chain reaction (PCR) using sets of primers were designed to include the upstream and downstream sequence of the gene based on DNA database found in NCBI GenBank.

Agarose gel electrophoresis indicated that the approximate molecular weight of the gene was 1.65 kb for both isolates.

The groEL gene of the two isolates was partially sequenced and compared with the gene sequence of NCBI homologous sequence from the GenBank Library of S.

Typhimurium LT2.

Such comparison revealed homogeneity of 100% between the sequenced part of the gene by RTSF and the sequence of GenBank.

Equally important, the sequence homology between the two isolated genes was also assessed and the results demonstrated 100% homology between the sequenced regions.

The purified groEL gene was inserted into the expression vector pBADTOPO in frame with 6-histidine tag sequence at C terminal end.

The derivative named pBAD-groEL was then introduced into chemically competent E.

coli TOP10 by transformation, all the tested clones for the presence of the recombinant vector shown positive result.

Recombinant vector was checked for correct orientation insertion of the gene into the vector by using PCR test, double digestion with NcoI and PmeI restriction enzymes to obtain the gene from the vector.

Agarose gel electrophoresis of the results of the two tests demonstrated that the gene was inserted in a correct orientation into the vector in four clones out of six tested clones of E.

coli harboring the groEL gene of S.

Typhimurium (LT2) ATCC 19585; named as ATCC (3, 4, 6 and 8) clones, and one clone from the three tested clones of E.

coli harboring the groEL gene of S.

Typhimurium DT104; named DT104 (2) clone.

One clone of each (E.

coli ATCC (3) and E.

coli DT104 (2)) was Summary II sequenced to confirm the previous results for correct insertion of the gene into the vector.

Results of sequencing showed that the genes were correctly inserted into the vector in both clones.

To express the recombinant HSP60 (GroEL), transformed E.

coli was grown and induced by L-arabinose.

The expressed recombinant protein was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

The optimum conditions for the recombinant protein expression were studied, by using different concentration of L-arabinose (0.2, 0.02, 0.002, 0.0002 and 0.00002%) and the optimum time for the production (1, 2, 3, 4, 5 and 6 hours) was also determined at 37°C.

It was observed that the optimum L-arabinose concentration was 0.02% after 4 hours incubation at 37°C.

SDS-PAGE analysis showed that the obtained band of protein on 10% SDS-PAGE was with an expected molecular weight of approximately 65-67 kDa as compared with the non-induced clone with Larabinose.

Finally, the protein was purified using Ni-NTA affinity chromatography column under denaturing conditions, which depicted high purity of it as detected on 10% SDS-PAGE gel.

To study the immunogenicity efficacy of the produced recombinant GroEL protein of S.

Typhimurium (LT2) ATCC 19585, groups of BALB/c mice were immunized subcutaneously with 30 μg GroEL, emulsified in complete Freund’s adjuvant.

Subsequent booster doses were given at 3 weeks intervals with GroEL protein emulsified in incomplete Freund’s adjuvant.

Antibody titers in sera of immunized mice were determined by ELISA method, and the results demonstrated a significant increase in anti-HSP antibody titers by the end of the 10th week as compared with control mice, in which no anti-HSP antibody was detected.

This suggests that the adjuvanted HSP had a good immunogenic property in mice.

To proceed further, groups of immunized mice were challenged with a lethal dose of S.

Typhimurium (LT2) ATCC 19585.

The overall survival rate of vaccinated mice suggests the protective value of the purified recombinant GroEL protein; an observation that may suggest the possible use of GroEL as a vaccine candidate in preventing typhoid and salmonellosis in human and animals.

التخصصات الرئيسية

الأحياء

الموضوعات

• حيوانات المختبر

عدد الصفحات

180

قائمة المحتويات

• APA
• MLA
• AMA

لغة النص

الإنجليزية

نوع البيانات

رسائل جامعية

رقم السجل

BIM-600885