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Soluble secretory expression of PG II-MBP fusion protein and application thereof

A fusion protein, MBP-PGII technology, applied in the field of soluble secretory expression and preparation of recombinant PG II fusion protein, can solve the problems of difficult industrialization, limited sources, high cost, etc.

Inactive Publication Date: 2015-10-07
BEIJING JIAWAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, human pepsinogen is mainly extracted from human stomach tissue, the source is limited, the cost is high, and it is difficult to realize industrialization, so the recombinant expression of highly active pepsinogen has a good market prospect

Method used

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  • Soluble secretory expression of PG II-MBP fusion protein and application thereof
  • Soluble secretory expression of PG II-MBP fusion protein and application thereof
  • Soluble secretory expression of PG II-MBP fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Amplification of PG II coding sequence

[0015] Use the PCR method to amplify the coding sequence of PG II from the plasmid PET32a-PG II (constructed by our company). The 5' end of the primer PGC5 used has added the restriction site of BamH I, and the 5' end of the primer PGC3 has added HindIII enzyme cleavage site.

[0016] PGC5: 5'-aagggatccggtagcggctctggctcgggttctggcgcagttgtcaaggttcctttgaag-3'

[0017] PGC3: 5'-aagaagcttttagtggtggtggtggtggtggtggtgagcagcggtagcaaatccgac-3'

[0018] 1 μl of La Taq DNA polymerase (TaKaRa), 4 μl of 10 μM PGC5 and 4 μl of PGC3, 8 μl of 2.5 mM dNTP, and 10 μl of 10×PCR reaction buffer were added to 100 μl of the PCR reaction system. The reaction conditions of PCR were: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 1 minute and 30 seconds, and then back to pre-denaturation for a total of 25 cycles, with a final extension of 7 minutes. The tar...

Embodiment 2

[0019] Embodiment 2: Construction of MBP-PG II fusion protein expression vector

[0020] Take 2 μg of the PG II coding sequence obtained by PCR amplification in Example 1, and establish a 50 μl double enzyme digestion system, specifically as follows: 2 μg of the PG II coding sequence, 5 μl of 10×K enzyme digestion reaction buffer, 1.5 μl of BamH I and Add 1.5 μl of Hind III to a total volume of 50 μl with double distilled water, and react in a water bath at 37°C for 6 hours. The double digestion product of the PG II coding sequence was purified using a gel extraction kit (OMEGA BIO-TEK).

[0021] Take 600ng of pMAL-p2X (NEB Company) vector, and set up 50 μl of double enzyme digestion system, as follows: 600ng of pMAL-p2X vector, 5 μl of 10×K digestion reaction buffer, 1.5 μl of BamH I and 1.5 μl of HindIII, add double-distilled water to a total volume of 50 μl, and react in a 37°C water bath for 6 hours. The pMAL-p2X vector double digestion product was purified using a gel e...

Embodiment 3

[0024] Embodiment 3: Expression of MBP-PG II fusion protein

[0025] The obtained pMAL-PG II plasmid was transformed into E.coli BL21(DE3) (Novagen Company) competent cells. Induced expression of bacteria: the clones were picked and inoculated in 100 ml LB liquid medium containing 100 μg / ml ampicillin, cultured overnight at 37° C. and 250 rpm to prepare seed solution.

[0026] Take 10ml of seed solution and inoculate into a 3L Erlenmeyer flask filled with 1L LB-Amp medium, and culture at 37°C until OD 600 0.5, then add IPTG with a final concentration of 0.5mM, lower the temperature to 28°C for 8 hours, and then centrifuge at 12000g to obtain the bacteria. Whole bacteria electrophoresis samples were prepared, and protein electrophoresis was used to analyze the expression of the target protein. The sample preparation method, electrophoresis voltage, gel staining and decolorization methods for SDS-PAGE electrophoresis are described in the second edition of "Molecular Cloning Ex...

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Abstract

The invention belongs to the technical field of bioengineering and particularly relates to soluble secretory expression of a fusion protein of a human pepsinogen II (PG II) and a maltose binding protein (MBP) in escherichia coli, provides a preparation method of the fusion protein, and also discloses application of a recombinant BMP-PG II fusion protein to preparation of monoclonal antibodies and calibration materials of pepsinogen II kits. Through the adoption of elements such as a Ptac promoter, an malE signal peptide and the maltose-binding protein, the soluble secretory expression of PG II from periphery to inside of escherichia coli is implemented; the immunogen activity of the recombinant human PG II protein from a prokaryotic expression system is greatly improved; and the preparation cost of the recombinant PG II is effectively reduced.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a soluble secreted expression of fusion protein of recombinant PG II and its preparation method and application. Background technique [0002] Pepsinogen (PG) is the enzyme precursor of pepsin in gastric juice, which is converted into active pepsin under acidic conditions of pH 1-5. According to its biochemical characteristics and immunogenicity, it can be divided into 2 subgroups, the immunogenicity of components 1-5 is the same, called PGI (also known as PGA), mainly composed of the principal cells and mucous neck cells of the fundic gland Secretion; components 6 and 7 are called PG II (also known as PGC), in addition to being secreted by the principal cells and mucous neck cells of the fundic glands, mucous neck cells of the cardia glands and pyloric glands of the gastric antrum, and duodenal The upper segment also produces PG II. [0003] Under normal cir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K19/00C07K16/40G01N33/573G01N33/577C12R1/19
Inventor 孔毅荣于海双李小红
Owner BEIJING JIAWAN BIOTECH CO LTD
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