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Cloning and Expression of Rat Proinsulin Coding Sequence

Author: ShuJianBo
Tutor: GuoGang
School: Tianjin Medical University
Course: Biochemistry and Molecular Biology
Keywords: Insulin Fusion expression Affinity chromatography High density fermentation
CLC: Q784
Type: Master's thesis
Year: 2008
Downloads: 20
Quote: 0
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Abstract


Diabetes is a common disease caused by the interaction of genetic and environmental factors , but currently in the process to study the pathogenesis of diabetes , lack of insulin to replace animal insulin as research reagents , only human , causing a lot of problems . Therefore, the cloning and expression of the rat insulin has important application value . We use gene engineering method cloned rat proinsulin gene coding sequences for efficient expression in E. coli . We extracted total RNA of rat pancreatic tissue , and reverse transcribed into cDNA amplification rat insulin gene encoding exon sequences cloned into pGEM - T Easy vector . Sequencing confirmed the correct sequence of the target gene insert converged prokaryotic expression vector pQE - 30XA , was transformed into E. coli M15 [ pREP4 ] fusion expression . SDS-PAGE and view the expression product size and the expression of Western - blotting further identification . The recombinant plasmid pQE-30Xa/proinsulin , optimize the expression condition expression of a large number of the Proinsulin the expression level of 19 mg / L medium . The recombinant protein proinsulin accounted for 28.24% of the bacterial protein . Target protein with 6 × His purification tag can be adsorbed on the metal chelate affinity chromatography Ni2 - IDA resin chromatography column , washed to remove non-specific adsorption of contaminating proteins , the target protein elution , the use of the Ni2 _IDA protein purification layer analysis column on the product of the expression of protein purification . The purified protein was digested , the digested product was again purified using Ni2 column , and the identification of the Tricine - SDS - PAGE to give a mature insulin . Enzyme-linked immunosorbent assay , the results showed a good activity . Preliminary exploration of the high-density fermentation in E. coli , wet bacteria 26g / L .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering) > The recombinant
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