CN104962573A - Soluble secretory expression of PG II-MBP fusion protein and application thereof - Google Patents

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

The solubility secreting, expressing of PG II and MBP fusion rotein and application thereof

Technical field

The invention belongs to technical field of bioengineering, be specifically related to fusion rotein solubility secreting, expressing of a kind of PG II that recombinates and its preparation method and application.

Background technology

Propepsin (Pepsinogen, PG) is pepsic enzyme precursor in gastric juice, changes into activated stomach en-under the acidic conditions of pH 1-5.Can be divided into 2 subgroups according to its biochemical characteristic and immunogenicity, the immunogenicity of component 1-5 is identical, is called PGI (also known as PGA), primarily of chief cell and the secretion of mucus neck cell of fundic gland; Component 6 and 7 is by title PG II (being also called PGC), and except being secreted by the chief cell of fundic gland and mucus neck cell, mucus neck cell and the duodenum epimere of the pyloric gland of cardiac gland and stomach hole also can produce PG II.

Under normal circumstances, the propepsin major part that stomach mucous membrane produces enters gastral cavity, and the postactivated one-tenth stomach en-of acidifying, digests protein, about has the PG of 1% to enter blood circulation through gastric mucosa capillary vessel.Serum PG I and PG II reflects the quantity of gastric mucosa body of gland and cell, also indirectly reflects the secreting function of gastric mucosa different sites.When gastric mucosa generation pathological change, serum PG content also changes thereupon.Therefore, the concentration of monitoring PG in serum can as the means of monitoring gastric mucosa state.

Serum PG level reflects the morphology and function of different sites stomach mucous membrane: PGI is the pointer detecting oxyntic gland cell function, and gastric acid secretion increases PGI and raises, and secretion reduces or stomach mucous membrane body of gland atrophy PGI reduces; The dependency comparatively large (relative to antrum) of PG II and gastric mucosa pathology, it raises and rises in value relevant with fundic gland shrink tube, gastric metaplasia or Pseudopyloric gland metaplasia, abnormal shape; The reduction of PGI/II ratio Progressive symmetric erythrokeratodermia is in progress relevant to atrophy of gastric mucosa.Therefore simultaneous determination PGI and PG II ratio can play the effect of fundic gland mucous membrane " serology rifle alive ".

The single chain polypeptide of propepsin to be molecular weight be 42KDa, containing 3 continuous print disulfide linkage, in prokaryotic expression system, intracellular expression is difficult to correct folding, and albumen exists with the inclusion bodies of non-activity, and after renaturation, activity is very low.It is thoughtful that the present invention utilizes malE natural signals peptide that PG II is guided to intestinal bacteria, achieve the solubility secreting, expressing of PG II albumen, recombinant expressed PG II albumen has good immunogenicity, the N end of the PG II albumen in the present invention has merged MBP albumen, and it further improves the characteristic of the unstable easily degraded of PG II albumen while improving PG II fusion protein expression.

At present, human pepsinogen mainly extracts from people's stomach-tissue, and limited source is with high costs, is difficult to realize industrialization, so recombinant expressed highly active propepsin has good market outlook.

Summary of the invention

The present invention discloses a kind of method utilizing escherichia expression system excreting and expressing recombinant human PG II fusion rotein; Additionally provide the preparation method of this fusion rotein simultaneously.

The invention also discloses and use the PG II fusion rotein of aforesaid method acquisition as immunogen in the monoclonal antibody of preparation PG II, and the application in propepsin diagnostic reagent.

Accompanying drawing explanation

Fig. 1. the recombinant plasmid vector design of graphics comprising PG II gene of the present invention;

Fig. 2. the Expression and purification SDS-PAGE electrophorogram of recombinant expressed PG II fusion rotein of the present invention;

Fig. 3. the SDS-PAGE electrophorogram that the fusion rotein that purifying of the present invention obtains is cut through Factor Xa enzyme;

Fig. 4. fusion rotein prepared by the present invention is tired as the PG II monoclonal antibody prepared by immunogen;

Fig. 5. the PG II Activity determination of fusion rotein prepared by the present invention.

Embodiment

The amplification of embodiment 1:PG II encoding sequence

Use PCR method to amplify the encoding sequence of PG II from plasmid PET32a-PG II (our company's structure), the 5 ' end of primer PGC5 used with the addition of the restriction enzyme site of BamH I, and the 5 ' end of primer PGC3 with the addition of the restriction enzyme site of HindIII.

PGC5：5’-aagggatccggtagcggctctggctcgggttctggcgcagttgtcaaggttcctttgaag-3’

PGC3：5’-aagaagcttttagtggtggtggtggtggtggtggtgagcagcggtagcaaatccgac-3’

Add the La Taq archaeal dna polymerase (TaKaRa) of 1 μ l in the PCR reaction system of 100 μ l, the dNTP of PGC5 and PGC3 each 4 μ l, the 2.5mM of 10 μMs adds 8 μ l, and 10 × PCR reaction buffer adds 10 μ l.The reaction conditions of PCR is: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 30 seconds, and 57 DEG C of annealing 30 seconds, 72 DEG C extend 1 minute 30 seconds, then get back to denaturation, carry out 25 circulations altogether, finally extension 7 minutes.Be separated the object band in PCR primer by agarose gel electrophoresis, then cut by object band, colloidal sol reclaims.

The structure of embodiment 2:MBP-PG II fusion protein expression vector

Get the PG II encoding sequence that in 2 μ g embodiments 1, pcr amplification obtains, set up the double digestion system of 50 μ l, specific as follows: the PG II encoding sequence of 2 μ g, 10 × K endonuclease reaction damping fluid of 5 μ l, the Hind III of 1.5 μ l BamH I and 1.5 μ l, add distilled water to cumulative volume 50 μ l, react 6 hours in 37 DEG C of water-baths.Gel is used to reclaim the double digestion product of test kit (OMEGA BIO-TEK) purifying PG II encoding sequence.

Get pMAL-p2X (NEB company) carrier of 600ng, set up the double digestion system of 50 μ l, specific as follows: the pMAL-p2X carrier of 600ng, 10 × K endonuclease reaction damping fluid of 5 μ l, 1.5 μ l BamH I with the HindIII of 1.5 μ l, add distilled water to cumulative volume 50 μ l, react 6 hours in 37 DEG C of water-baths.Gel is used to reclaim test kit (OMEGA BIO-TEK) purifying pMAL-p2X carrier double digestion product.

Ligation: BamH I and the HindIII double digestion product of getting the purified pMAL-p2X of 4 μ l, add the double digestion product of the purified PG II of 6 μ l, the T4 DNA ligase (TaKaRa) of 1 μ l and 10 × T4 DNA ligase reaction buffer of 1 μ l, connect in 16 DEG C of water-baths and spend the night.

Connect product conversion bacillus coli DH 5 alpha, then picked clones, (preparation method of competence DH5 α and method for transformation are according to described in " Molecular Cloning: A Laboratory guide " second edition to send company to check order.J. Pehanorm Brooker work).Gained recombinant plasmid called after pMAL-PG II.

The expression of embodiment 3:MBP-PG II fusion rotein

By obtained pMAL-PG II Plastid transformation E.coli BL21 (DE3) (Novagen company) competent cell.The abduction delivering of bacterium: picked clones is inoculated in 100ml containing in the LB liquid nutrient medium of 100 μ g/ml penbritins, 37 DEG C, overnight incubation under 250rpm condition, preparation seed liquor.

Get 10ml seed liquor and inoculate 3L Erlenmeyer flask into being equipped with 1L LB-Amp substratum, 37 DEG C are cultured to OD ₆₀₀be about 0.5, then add the IPTG that final concentration is 0.5mM, after temperature being reduced to 28 DEG C of induction 8h, the centrifugal acquisition thalline of 12000g.Prepare full bacterium electrophoresis Sample, protein electrophoresis analysis purposes protein expression situation.The sample preparation methods of SDS-PAGE electrophoresis, electrophoretic voltage, method that is gel-colored and decolouring are shown in described in " Molecular Cloning: A Laboratory guide " second edition.J. Pehanorm Brooker work.

Embodiment 4: the colibacillary thoughtful Protein Extraction containing MBP-PG II gene

Hypertonic solution process thalline: add 1/10 bacteria liquid and amass high sepage (20mM Tris, 20% (W/V) sucrose, 1mM EDTA, pH8.0), resuspended thalline, places 10 minutes, centrifugal 15 minutes of 12000g on ice, collects supernatant;

Hypotonic dope process thalline: add 1/10 bacteria liquid and amass hypotonic medium (0.1mM MgCl ₂), resuspended thalline, places 10 minutes, centrifugal 15 minutes of 12000g on ice, collects supernatant.

The purifying of embodiment 5:MBP-PG II fusion rotein

1) QFF purifying protein: the high and low treatment solution that oozes mixes rear loading, with Elution Buffer (20mM Tris, the 1M NaCl) eluted protein of 5-10 column volume;

2) HiTrap Chelating HP (nickel ion chelating affinity column, GE) purifying protein: after loading, first use Wash Buffer (20mM Tris, 0.5M NaCl, 80mM imidazole, pH8.0) foreign protein is rinsed out, then Elution Buffer (20mM Tris, 0.5M NaCl, 300mM imidazole, pH8.0) wash-out target protein is used.

Embodiment 6:MBP-PG II fusion protease cuts qualification

The MBP-PG II fusion rotein of preparation has the restriction enzyme site of Factor Xa, with Factor Xa, enzyme is carried out to the fusion rotein obtained and cut qualification, concrete enzymatic cleavage methods: MBP-PG II fusion rotein and Factor Xa mass ratio are 50: 1, enzyme cutting buffering liquid (20mM Tris, 100mM NaCl, 2mM CaCl ₂, pH8.0), 23 DEG C, enzyme cuts 6h.BMP label protein can be removed with aforesaid method, prepare the PG II recombinant protein of natural size.

Embodiment 7:MBP-PG II fusion protein immunization mouse, preparation monoclonal antibody

1) immune mouse: the restructuring MBP-PG II fusion rotein of preparation mixes emulsification, subcutaneous inoculation by every mouse 100 μ g protein content and Freund's complete adjuvant equal-volume; After 3 weeks, second immunisation, 50 μ g protein contents and Freund's incomplete adjuvant equal-volume mix, after emulsification, abdominal injection immunity; After 2 weeks, three immunity, the same second immunisation of method; After 7 days, adopt tail vein and detect, tire and be greater than 24W.

2) monoclonal antibody preparation: get immune mouse spleen cell and murine myeloma cell SP2/0 merges, carry out the cultivation of HAT selectivity, ELISA method is utilized to filter out positive cell strain, carry out three mono-clonalizations to cultivate, obtain the monoclonal cell strain of stably excreting antibody, after enlarged culturing is carried out in the strain of every strain monoclonal antibody hybridoma cell, preparation ascites.

3) antibody purification and bioactivity: the ascites of collection, through protein A column purification, obtains high-purity mouse monoclonal albumen.Wrap respectively by 5ug/ml MBP-PG II fusion rotein and MBP albumen, the ascites antibody utilizing ELISA to detect preparation is tired.

Embodiment 8:MBP-PG II fusion protein immunization originality detects

MBP-PG II fusion rotein 340ng/ml is initial detectable level, continuously half-and-half dilution, with the PG II content of the quantitative different concns fusion rotein of PG II immunoturbidimetry reagent (the last nine biotech firm).

Claims (8)

1. the structure of the prokaryotic expression plasmid of a people PG II fusion rotein, it is characterized in that the gene of PG II to import E. coli expression strains by expression plasmid pMAL-p2X or pMAL-p5X, IPTG induce the secretion of PG II fusion rotein to colibacillary thoughtful in thus obtain solubility expression.

2. expression plasmid according to claim 1, is characterized in that plasmid comprises the sequence of Ptac promotor-maleE signal peptide-malE gene-FactorXa restriction enzyme site-PG II gene-8 × His Tag.

3. one kind utilizes the fusion rotein of the MBP-PG II in source, the thoughtful chamber of the expression of recombinant plasmid intestinal bacteria described in claim 1-2, it is characterized in that No. genebank of PG II for J04443.1, fusion rotein has SEQ ID No 1 aminoacid sequence in sequence table; Or by the amino-acid residue of fusion rotein under the prerequisite not changing fusion rotein characteristic, carry out the replacement of partial amino-acid, disappearance or add institute obtaining recombinant protein.

4., containing maleE albumen natural signals peptide in the expression vector according to claim 1-3, for the secreting, expressing of MBP-PG II, it is characterized in that signal peptide has SEQ ID No 2 aminoacid sequence in sequence table.

5. the recombinant expressed PG II fusion rotein according to claim 1-4, its N holds and merges bmp protein, in order to improve fusion protein expression and stable fusion rotein.

6. the Host Strains utilizing the expression plasmid described in any one of claim 1-5 to import expression comprises following colibacillus engineering strain: DH5 α, TOP10, TB1 and BL21.

7. recombinant soluble described in claim 1-6 expresses a preparation method for the fusion rotein of PG II, it is characterized in that, comprises the following steps:

(1) bacterial strain of high expression MBP-PG II fusion rotein is screened;

(2) positive strain is used LB culture medium culturing, IPTG induction obtains the bacterium liquid containing MBP-PG II fusion rotein;

(3) collected by centrifugation thalline, high hypotonic solution process thalline, the thoughtful albumen of extracting;

(4) anion-exchange chromatography post and metal ion-chelant affinity column purified fusion protein.

8. the PG II fusion rotein for preparing of claim 1-7 in the monoclonal antibody of preparation PG II as an immunogen, and the application in propepsin diagnostic reagent.