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Human pepsinogen I (PGI)-maltose-binding protein (MBP) fusion protein soluble secretory expression and use

A fusion protein, MBP-PGI technology, applied in the field of soluble secretory expression of recombinant PG I fusion protein and its preparation, can solve the problems of difficulty in correct folding, low activity, and difficulty in industrialization, etc.

Inactive Publication Date: 2015-10-28
BEIJING JIAWAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, human pepsinogen is mainly extracted from human gastric tissue. This traditional extraction method has a low product yield and is limited by limited material sources. The cost is high and it is difficult to achieve industrialization. Therefore, recombinant expression of highly active gastric Proteaseogen has a good market prospect
[0006] Pepsinogen is a single-chain polypeptide with a molecular weight of 42KDa, which contains 3 consecutive disulfide bonds. It is difficult to fold correctly in intracellular expression in a prokaryotic expression system. The protein exists in the form of inactive inclusion bodies, and its activity is very low after renaturation.

Method used

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  • Human pepsinogen I (PGI)-maltose-binding protein (MBP) fusion protein soluble secretory expression and use
  • Human pepsinogen I (PGI)-maltose-binding protein (MBP) fusion protein soluble secretory expression and use
  • Human pepsinogen I (PGI)-maltose-binding protein (MBP) fusion protein soluble secretory expression and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Amplification of PG I Coding Sequence

[0014] Use the PCR method to amplify the coding sequence of PG I from the plasmid PET32a-PG I (constructed by our company). The 5' end of the primer PGA35 used has added the restriction site of Xba I, and the 5' end of the primer PGA33 has added HindIII enzyme cleavage site.

[0015] PGA35: 5'-AACTCTAGAGGTAGCGGCTCTGGCTCGGGTTCTATTATGTATAAAGTTCCATTGATTAGAAGAAG-3'

[0016] PGA33: 5'-AACAAGCTTTTAGTGGTGGTGGTGGTGGTGGTGGTGGGCGACTGGTGCCAAACC-3'

[0017] 1 μl of La Taq DNA polymerase (TaKaRa), 4 μl of 10 μM PGA35 and 4 μl of PGA33, 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 56°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...

Embodiment 2

[0018] Embodiment 2: Construction of MBP-PG I fusion protein expression vector

[0019] Take 2 μg of the PG I 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 I coding sequence, 5 μl of 10×M digestion reaction buffer, 1.5 μl of Xba I and Add 1.5 μl of HindIII 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 I coding sequence was purified using a gel extraction kit (OMEGA BIO-TEK).

[0020] 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×M digestion reaction buffer, 1.5 μl of Xba 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 extraction kit ...

Embodiment 3

[0023] Embodiment 3: the expression of MBP-PG I fusion protein

[0024] The obtained pMAL-PG I 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.

[0025] 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 1PTG 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 ...

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Abstract

The invention belongs to the technical field of bioengineering, relates to soluble secretory expression of a human pepsinogen I (PGI)-maltose-binding protein (MBP) fusion protein in escherichia coli and provides a preparation method of the fusion protein. The invention also discloses a use of the recombinant BMP-PGI fusion protein in preparation of a monoclonal antibody and a pepsinogen I kit calibrator. Through use of a Ptas promoter, a malE signal peptide and a maltose binding protein, soluble secretory expression of PGI in escherichia coli is realized so that immunogen activity of the recombinant PGI protein of a prokaryotic expression system is greatly improved and a recombinant PGI preparation cost 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 I and its preparation method and application. Background technique [0002] Gastric cancer is one of the most common malignant tumors. Its morbidity and fatality rate have always been the top among malignant tumors in my country. Early diagnosis and early interventional treatment are one of the key measures to reduce the fatality rate. Recent studies have found that serum pepsinogen (PG) can be used as a serum screening index for gastric cancer and precancerous lesions. [0003] Pepsinogen (Pepsinogen, PG) is the non-enzyme-active precursor of pepsin, which is converted into active pepsin under the acidic condition of pH 1-5. PG has 7 isozymes, which can be divided into PG I subgroup (also known as PGA) and PG II subgroup (also known as PGC) according to their biochemical immune characteristics....

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K19/00C12N9/64C07K14/245C12N1/21C07K16/40G01N33/573
Inventor 孔毅荣于海双李小红
Owner BEIJING JIAWAN BIOTECH CO LTD
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