Cloning and expression of somatotropin gene from local cows in Basrah
Other Title(s)
تنسيل و تعبير جين السوماتوتروبين من الأبقار المحلية في البصرة
Dissertant
al-Salam, Rasha Mundhir Uthman
Thesis advisor
University
University of Basrah
Faculty
Science College
Department
Department of Biology
University Country
Iraq
Degree
Ph.D.
Degree Date
2012
English Abstract
The present study, have undertaken the cloning of bovine growth hormone or bovine somatotropin gene from local cows breeds in Basrah city, and optimizing the conditions for its high-level expression in Escherchia coli.
Furthermore, cost effective refolding and purification strategies to obtain a biologically active bovine somatotropin were investigated too.
To achieve this goal the ribonucleic acid RNA extraction from whole cow blood and used as a template for the synthesis of bovine growth hormone cDNA by RT-PCR, through using specific primers.
The 677 bp RT-PCR amplified product was inserted in to the Pst1 site of pBR322 via the poly dC : poly dG joining technique to produce the recombinant vector.
The cloning vector then transformed in to E.coli HB101 and the trans formant cells colonies were selected by tetracycline Resistant / ampicillin sensitive phenotype .The efficiency transformation was also determined to be 2 × 107 cfu / μg.
The fermentation strategy for high cells density growth of E.coli harboring the bST gene was carried out using shake flask cultivation in LB media supported with 2 % glucose at 37º C.
The highest cells density level was determined to be 1.540 at OD600 after 8 hs of cells growing.
Since, the eukaryotic gene was expressed as insoluble aggregates called inclusion bodies ; so conditions were optimized for isolation, solubilization and purification of The bST hormone from the inclusion bodies in bacterial cells and resulting in a full refolded protein form indicated by 12 % SDS-Polyacrylamide gel electrophoresis.
Followed by purification the hormone using DEAES epharoseion- exchange chromatography and the result was a single visible band with 22KDa on 12 % SDS-Polyachrylamid gel which represents the purified bST.
The activity of the purified bST was studied using twenty 8-10 weeks old inbred Albino, Mus muscles L, Balb / C mice which treated with the purified bST to detect the immunological stability of bST, using the sandwich ELISA for this purpose.
The highest rate of the bST concentration level were observed in 5 treated mice 25 % with concentration 180 ng / ml followed by the rate of 15 % in 3 treated mice and with concentration 190 ng / ml.
Main Subjects
Topics
No. of Pages
170
Table of Contents
Table of contents.
Abstract.
Chapter One : introduction and literature review.
Chapter Two : materials and methods.
Chapter Three : results and discussion.
Conclusion and recommendation.
References.
American Psychological Association (APA)
al-Salam, Rasha Mundhir Uthman. (2012). Cloning and expression of somatotropin gene from local cows in Basrah. (Doctoral dissertations Theses and Dissertations Master). University of Basrah, Iraq
https://search.emarefa.net/detail/BIM-317401
Modern Language Association (MLA)
al-Salam, Rasha Mundhir Uthman. Cloning and expression of somatotropin gene from local cows in Basrah. (Doctoral dissertations Theses and Dissertations Master). University of Basrah. (2012).
https://search.emarefa.net/detail/BIM-317401
American Medical Association (AMA)
al-Salam, Rasha Mundhir Uthman. (2012). Cloning and expression of somatotropin gene from local cows in Basrah. (Doctoral dissertations Theses and Dissertations Master). University of Basrah, Iraq
https://search.emarefa.net/detail/BIM-317401
Language
English
Data Type
Arab Theses
Record ID
BIM-317401
