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Expression of human cardiac muscle type fatty acid binding proteins in bacillus coli DH5 alpha and purification

A fatty acid combination and Escherichia coli technology, applied in the biological field, can solve the problems of high price and low yield, and achieve the effect of avoiding denaturation and refolding process

Inactive Publication Date: 2008-08-27
侯玥 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But they also have disadvantages such as high price and low yield (Table 1)

Method used

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  • Expression of human cardiac muscle type fatty acid binding proteins in bacillus coli DH5 alpha and purification
  • Expression of human cardiac muscle type fatty acid binding proteins in bacillus coli DH5 alpha and purification
  • Expression of human cardiac muscle type fatty acid binding proteins in bacillus coli DH5 alpha and purification

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Experimental program
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Effect test

Embodiment 1

[0096] 1. Plasmids and strains

[0097] Escherichia coli (E.coli) DH5α strain, pGEM-T easy, PBV220 protein expression plasmid.

[0098] 2. Tool enzymes and biochemical reagents

[0099] Restriction endonuclease, T4 DNA ligase, Taq DNA polymerase, plasmid DNA extraction kit, DNA gel recovery kit, human H-FABP monoclonal antibody (Clone 6B6), horseradish peroxidase-labeled di Anti, RNAase inhibitor, AMV reverse transcriptase, Sephacryl S-100 HR, Sepharose G-75.

[0100] 3. Oligonucleotide primers

[0101] Following the principles of PCR primer design, according to the human H-FABP cDNA sequence of GenBank (NM004012), the 5' and 3' end oligonucleotide primers encoding the H-FABP gene fragment were designed, and EcoR was introduced at the respective 5' ends. I and Sal I restriction enzyme sites. The primer sequences are as follows:

[0102] Forward primer: 5'-gcg aat tca tgg tgg acg ctt tc-3';

[0103] Reverse primer: 5'-att gtc gac tca tgc ctc ttt ctc-3',

[0104] 4. RT-PC...

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Abstract

The invention relates to the expression and purification of human heart-type fatty acid-binding protein in escherichia coli DH5 alpha, which belongs to the biological technical field, the expression and purification comprises: firstly, preparing an oligonucleotide primer, secondly, amplifying with RT-PCR and determining a sequence, thirdly, constructing recombinant expression plasmids, fourthly, expressing heart-type fatty acid-binding protein through inducing, and fifthly, extracting and purifying the human heart-type fatty acid-binding protein. The expression and the purification of the invention have the beneficial effect that a PL promoter is enabled to enter a complete inhibitory state. H-FABP protein is expressed in PBV220 through utilizing the characteristic. We optimize the temperature and the time in the expression process, bacteria is increased under the temperature of 30DEG C, and high level expression is obtained through inducing for 5 hours under the temperature of 42 DEG C. The successful clone and expression of the human heart-type fatty acid-binding protein lay the foundation of further researching the significance of the heart-type fatty acid-binding protein in the diagnosis and therapeutic effect evaluating of diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology. Background technique [0002] 1. Heart-type fatty acid-binding protein (H-FABP) [0003] (1). The nature and characteristics of FABP [0004] Fatty Acid Binding Protein (FABP) was first discovered by Ockner et al. in 1972 when studying the regulation of fatty acid absorption in the small intestine of rats, a family of homologous small molecule intracellular proteins found in the intestinal mucosa [1] . The molecular weight is 12-16KD, widely distributed in mammalian small intestine, liver, fat, heart, brain, skeletal muscle and other cells [2] . The discovered FABPs include cardiac muscle type (H-FABP), small intestine type (I-FABP), liver type (L-FABP), adipocyte type (A-FABP), brain cell type (B-FABP), kidney type ( K-FABP), skeletal muscle type (s-FABP), psoriasis-associated type (PA-FABP) and epidermal type (E-FABP) 9 types [1-6] . FABPs are named after their distribution in tissues, and a...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/12C12N15/70C07K14/435
Inventor 侯玥许淑芬侯巍于源华张淑华
Owner 侯玥
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