Cloning of human insulin gene in bacterial expression system

Other Title(s)

هرمون الأنسولين البشري في النظام البكتيري

Dissertant

Salih, Afrudit Abd al-Razzaq

Thesis advisor

al-Badran, Adnan Isa
Mahdi, Kawthar Hawwaz

University

University of Basrah

Faculty

Science College

Department

Department of Biology

University Country

Iraq

Degree

Ph.D.

Degree Date

2012

English Abstract

Human insulin was produced using genetic engineering methods in bacterial expression system, under Iraqi conditions and the study of its treatment application. Total RNA samples were purified from fresh human blood by using a specific QIAamp RNA mini kit, the results showed that high purity of total RNA samples with two distinct bands represented the two main sub units of human total RNA which are 28S rRNA and 18S rRNA, with a high level of concentration reaching to 36.6 and 34.6μg \ ml.

at 260 nm. A first single strand cDNA was obtained by reverse transcription process from human total RNA using specific AMV reverse transcriptase enzyme. Human proinsulin gene was amplified from cDNA strand by using specific primers, and the result shows a fragment of about 1183 kb.

Which was obtained as a sharp band, then the gene was sub cloned into expression vector pET- Blue. 1- Vector, and a new constructive vector pET Blue- 1-hpi vector was achieved as a first time in the world with about 4659 kb the transformation processes were done directly to three types of genetic engineering bacterial cells which are : Nova Blue Singles™, Tuner (DE3) pLacI and E.

coli HB101 and the result shows the high levels of transformation for all types of strains which reached to (4 * 106, 52 * 106, 124 * 106) cfu \ ml., respectively. Luria Britain broth medium induced with IPTG (1mM), was used to determine the expression of human proinsulin and the results of 12 % SDS \ PAGE analysis show the presence of about 10 KDa of human proinsulin up to 50 % of the bacteria protein constituted by only Tuner (DE3) pLacI as insoluble protein (pellet), while the other two types of bacterial strains Nova Blue Singles™ and E.

coli HB101 do not express the hormone yet Lactose (1mM) was used as analogues inducer to enhance the expression of human proinsulin and the results show the high level of hormone expression with large amount of biomass and protein. Following the production of proinsulin inclusion bodies, these were solubilized in 8 M urea and refolded in a medium containing final concentration of 2M chemotropic reagent at 4Cº, pH 9.0 using 0.5 : 5 mM cystine : cysteine ratio, which gave the refolded proinsulin detected by 12 % PAGE analysis and purified by an ion exchange chromatography on DEAE Sepharose which gave distinct peaks of proteins with linear gradient of 0.8 M NaCl, and the proinsulin peak appeared in 0.2- 0.25 M NaCl after analysis with 12 % SDS \ PAGE which appeared as a sharp band with about 10 KDa The corrected refolded purified human insulin was converted to human insulin by the treatment with proteolytic solution at 37 C º for 30 min.

and the human insulin was subjected to G-75 sephadex chromatography for further purification and determines the molecular weight by using several types of standard proteins, and the result shows the expected size of human insulin which reached to 5850 KDa. Human proinsulin and insulin were chemically identified by FTIR, spectroscopy analysis, which show the main functional groups of the polypeptide hormones. The cytotoxicity of the human insulin was studied against fresh human blood cells and the result illustrates that there were not any cytotoxicity effect of the product against the blood cells within different concentrations ranging from 50 - 250 ppm. The immunoassay was also done in this study by using an ELISA test with a specific human insulin antibody and the results of the test confirm the characteristic feature of the product as human insulin and also determined the concentration of the product within its solution which reached to 6.20μU \ ml.

within dilution 1 : 10 of the sample The bioassay of the produced human insulin was also determined in this study against induced hyperglycemic domestic rabbits (Lepus cubiculum), and the statistically analysis of the results illustrated the highly significant (P > 100) reduction in glucose levels of diabetic rabbits groups which were treated with produced human insulin and commercially available standard human insulin (Elli Lilly and Co.), in comparison with non-treated animals, also in this study we demonstrated the effect of physical and chemical mutagens ( UV and NTG ), on the stability of the expressed cells, the vector and also to determine their ability for producing of human insulin by obtaining mutant strains, and that was achieved by getting the inclusion bodies of expressed human proinsulin as checked by microscope, and high levels of both bacterial biomass and proteins.

Main Subjects

Biology

Topics

• Freedom of expression
• Chemistry
• Biology
• Biotechnology
• Biochemistry
• Insulin
• Genes

No. of Pages

169

Table of Contents

• APA
• MLA
• AMA

Language

English

Data Type

Arab Theses

Record ID

BIM-317392