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Human bone morphogenic protein-4 mature peptide colibacillus high expression vector and its preparing method

A technology of Escherichia coli and expression vector, which is applied in the field of preparation of human bone morphogenic protein-4 mature peptide, can solve the problems of rejection, poor repeatability, complicated extraction steps, etc.

Inactive Publication Date: 2007-06-06
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Early BMP used in clinical treatment was obtained from fresh bone tissue, but there are many problems in extracting BMP from bone tissue: (1) The source of material is difficult: it is no problem to obtain a large amount of fresh animal bone tissue, but the extracted Animal BMP is immunogenic to humans and will cause rejection in the human body, so it is not suitable for use in the human body; it is very difficult to obtain a large amount of fresh human bone tissue
(3) The extraction steps are complicated: it takes a lot of manpower and time
(4) Low yield: generally less than 10mg of BMP can be extracted from 10kg of bone tissue, which is difficult to meet clinical needs
(5) The purity is not good: Although after many complicated extraction steps, the obtained BMP is still not pure, and there are still many impurity protein bands in electrophoresis analysis
(6) Poor reproducibility: the purity and biological activity of BMP obtained by each batch of extraction vary greatly, and it is difficult to have strict quality control in the production of BMP extracted from bone tissue
So far, there is no report on the construction of Escherichia coli genetic engineering high expression vector to produce human bone morphogenic protein-4 (rhBMP-4) mature peptide

Method used

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  • Human bone morphogenic protein-4 mature peptide colibacillus high expression vector and its preparing method
  • Human bone morphogenic protein-4 mature peptide colibacillus high expression vector and its preparing method
  • Human bone morphogenic protein-4 mature peptide colibacillus high expression vector and its preparing method

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Embodiment 1

[0186] The specific embodiment of the Escherichia coli high expression vector of human bone morphogenic protein-4 mature peptide is as follows:

[0187] (1) Total RNA was extracted from fresh human placenta tissue;

[0188] (2) cDNA library obtained by reverse transcription;

[0189] (3) Design and synthesize primers;

[0190] (4) The DNA sequence encoding human bone morphogenic protein-4 mature peptide was amplified from the cDNA library by PCR technology

[0191] (5) Cloning into the pDH2 vector constructed by our research group (see accompanying drawing 1);

[0192] (6) Obtain the pDH2-hB4m expression vector, carry out restriction enzyme digestion identification and DNA sequence determination identification (sequence see above)

[0193] (7) Transforming different E. coli strains, conducting induction expression and expression condition tests and high-density fermentation tests in strain fermenters;

[0194] The constructed expression vector pDH2-hB4m h...

Embodiment 2

[0196] Taking the 20040622 batch test as an example, the method for preparing rhBMP-4m with the expression vector pDH2-hB4m of the present invention is further described in conjunction with Figures 2 to 9, and the specific implementation steps are as follows:

[0197] Step 1: Transform E. coli host strain DH5α with the expression vector pDH2-hB4m:

[0198] Escherichia coli DH5α competent cells were transformed with pDH2-hB4m by conventional methods, and transformed bacteria pDH2-hB4m / DH5α were obtained through ampicillin resistance screening, identification of extracted plasmids, and detection of induced expression tests (see Figure 2). The transformed bacteria were cultured in shake flasks at 30°C to OD 600 After = 0.4 hours, induce the expression at 42°C for 1 hour, and the expression of the target protein (rhBMP-4m) can be seen. As the induction time prolongs, the amount of the target protein (rhBMP-4m) in the bacteria increases, reaches a plateau in 4 hours, and rea...

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Abstract

This invention relates to a colibacillus expression carrier of the 4 mature peptide of the protein formed by man-bone and a preparation method for the 4 mature peptide. Said carrier refers to the colibacilus expression carrier p DH2-hB4m got by recombining them with the molecular clone technology, the total length of the carrier is 4.014 nucleotide pairs. The preparation method includes determination steps of transforming expression culture, purification, renaturation and activity to the host bacteria.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to an Escherichia coli expression vector of human bone morphogenic protein-4 mature peptide and a preparation method of the expressed human bone morphogenic protein-4 mature peptide. Background technique [0002] (1) Overview of bone morphogenic proteins [0003] Urist in the United States first reported in 1965 that implanting bovine demineralized bone matrix into mouse muscles could induce the formation of new cartilage and bone tissue [1]. The generated active protein is named as bone morphogenetic protein (Bone Morphogenetic Protein, referred to as BMP), which theoretically confirms the theory of bone formation, and proposes a new approach for the treatment of clinical bone repair[2,3]. It was later found that BMP not only exists in animal and human bone and tooth tissues[4-9], but also in many tissues of embryonic and adult animals[10-11]. At least 20 different BMPs have...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/12C12P21/02C07K1/14A61P19/00
Inventor 陈苏民秦云陈南春
Owner FOURTH MILITARY MEDICAL UNIVERSITY