CN101225113B - Thymosin alpha1-thymopentin fusion peptide and preparation method thereof - Google Patents

Abstract

The invention discloses a thymosin Alpha 1 - thymopentin fusion peptide, which is characterized in that: amino acid sequence of the fusion peptide is shown as SEQ ID NO. 1; relative molecular mass is 4470 dalton; the thymosin Alpha 1 (T Alpha 1) gene and the thymopentin (TP5) gene are fused together and inserted into pichia expression vector pGAPZ Alpha A, thereby getting recombinant expression plasmid pGAPZ Alpha A/T Alpha 1-TP5; PYD culture medium is used to produce fusion protein before T Alpha 1-TP5 fusion peptide is get through thrombin digestion. The thymosin Alpha 1 - thymopentin fusion peptide is far longer than the thymopentin and the thymosin Alpha 1 in half life period of external plasma, and can obviously promote proliferation of spleen cells and keep immunoregulation activityof the thymopentin. The thymosin Alpha 1 - thymopentin fusion peptide has the advantages of long half life period, good activity and good application prospect compared with the thymopentin.

Description

A kind of thymosin alpha 1-thymopentin fusion peptide and preparation method thereof

Technical field

The present invention relates to a kind of fusogenic peptide and preparation thereof, relate in particular to a kind of Thymosin alpha L-thymopentin fusion (T α 1-TP5) and preparation method thereof, belong to biomedicine field.

Background technology

(Thymopentin is the amino-acid residue peptide section of the 32-36 of thymopoietin TP5) to thymopeptide-5, is a bidirectional immune regulator, now is used for the treatment of immunodeficient disease, autoimmune disease etc. clinically.Studies have shown that its transformation period is shorter, have only 30 seconds.The so short transformation period not only makes thymopeptide-5 short action time in vivo, and increases patient's drug cost, has influenced thymopeptide-5 effectiveness clinically.If can by certain means increase thymopeptide-5 stability, prolong in its body the transformation period and will improve the result of treatment of thymopeptide-5 greatly to reduce dosage or to prolong the administration cycle, reduce drug cost, promote its application clinically.

Thymosin alpha 1 (T α 1 claims thymosin again) extracts in thymus gland the earliest, is the polypeptide that is made of 28 amino-acid residues.Thymosin alpha 1 also is used for the treatment of disease of viral infection, immunodeficient disease, autoimmune disease, tumour etc. clinically as immunomodulator.

Can prolong the principle of transformation period in the small-molecular peptides body according to the prolongation of peptide chain, utilize genetic engineering technique that Thymosin alpha 1 and thymopeptide-5 are merged preparation thymosin alpha 1-thymopentin fusion peptide (T α 1-TP5), can prolong the transformation period of Thymosin alpha 1 and thymopeptide-5, and bring into play both immunoregulation effects simultaneously, to improve curative effect.By retrieval, the relevant paper and the patent of this research yet there are no report.

Summary of the invention

At short deficiency of thymopeptide-5 transformation period, the problem to be solved in the present invention provides a kind of thymosin alpha 1-thymopentin fusion peptide (T α 1-TP5) and preparation method thereof.

Thymosin alpha 1-thymopentin fusion peptide of the present invention (T α 1-TP5), be to be built into expression vector pGAPZ α A/T α 1-TP5 by genetically engineered by T α 1-TP5 fusion gene, after steps such as fermentation, isolation and purification, prepare again, be 140min the T α 1-TP5 fusogenic peptide external after measured plasma half-life that obtains, and compares the thymopeptide-5 transformation period obviously to prolong.

Thymosin alpha 1-thymopentin fusion peptide of the present invention (T α 1-TP5), it is characterized in that: the aminoacid sequence of described fusogenic peptide is shown in SEQIDNO.1, its relative molecular mass is about 4470 dalton, is that 3208 daltonian Thymosin alpha 1s and relative molecular mass are that 679.77 daltonian thymopeptide-5s fusions form by relative molecular mass.Or: described fusogenic peptide be comprise aminoacid sequence shown in the SEQ ID NO.1, molecular weight is less than 10000 daltonian peptide or protein.

The preparation method of thymosin alpha 1-thymopentin fusion peptide of the present invention (T α 1-TP5), be made up of following steps:

(1) structure of expression vector pGAPZ α A/T α 1-TP5

T α 1-TP5 fusion gene increases by the PCR system, and wherein, the leading primer of design contains EcoR I site, and the postorder primer contains Not I and zymoplasm site; T α 1-TP5 fusion gene behind plasmid pGAPZ α A and the purifying is all used EcoR I and Not I double digestion, agarose gel electrophoresis reclaims carrier and the T α 1-TP5 fusion gene segment after enzyme is cut, connect 16h with 4 ℃ of T4 dna ligases and be built into expression vector pGAPZ α A/T α 1-TP5, the transformed competence colibacillus intestinal bacteria, the picking positive colony is identified recombinant plasmid with DNA electrophoresis and sequential analysis.

(2) contain the structure of expression vector pGAPZ α A/T α 1-TP5 engineering bacteria

Expression vector pGAPZ α A/T α 1-TP5 transforms pichia spp GS115 competent cell after the Bln enzyme is cut, make up the engineering bacteria that contains expression vector pGAPZ α A/T α 1-TP5; Positive colony is 1000 μ gmL with containing concentration ^-1The PYD screening of microbiotic Zeocin; Adopt round pcr to identify the T α 1-TP5 fusion gene of positive colony, identify purpose T α 1-TP5 fusogenic peptide with electrophoresis and mass spectroscopy.

(3) expression of T α 1-TP5 fusogenic peptide with separate and identify

Under 30～32 ℃ of conditions, fermentation culture is 4～5 days in the PYD substratum with the pichia spp GS115-pGAPZ α A/T α 1-TP5 engineering bacteria that makes up; Centrifugal removal yeast cell, nutrient solution separates T α 1-TP5 fusogenic peptide with the nickel ion affinity chromatography, and identifies with electrophoresis; Isolating T α 1-TP5 fusogenic peptide further separates the T α 1-TP5 fusogenic peptide that obtains purifying with Sephadex G-50 after the zymoplasm enzyme is cut.

Wherein:

Described nickel ion affinity chromatography adopts nickel ion affinity chromatography system, comprising:

Binding buffer liquid: 20mmol/L Na ₂HPO ₄, 0.5mol/L NaCl, 20mmol/L imidazoles, pH7.4;

Elution buffer: 20mmol/L Na ₂HPO ₄, 0.5mol/LNaCl, 0.5mol/L imidazoles, pH7.4;

The desalination system of T α 1-TP5 fusogenic peptide: Tris damping fluid, 0.02mol/LTris alkali, 0.001mol/LEDTA, pH7.4;

Described zymoplasm enzyme is cut and is adopted the zymoplasm enzyme to cut system: 50mmol/LNH ₄HCO ₃Solution, pH8.4,28 ℃ of 4h;

Described Sephadex G-50 elutriant is: 50mmol/L (NH4) ₂SO ₄, pH8.0.

Be 140min thymosin alpha 1-thymopentin fusion peptide (T α 1-TP5) its external plasma half-life of using that the inventive method obtains, proves that by the proliferation experiment to splenocyte T α 1-TP5 fusogenic peptide has significant promotion kunming mice splenocyte proliferation activity.

Concrete activity research method of proof is as follows:

1.T α l-TP5 fusogenic peptide is measured external plasma half-life

Draw by HPLC mensuration, measuring method is made up of following steps:

(1) chromatographic condition

Chromatographic column: Shim-packVP-ODS C ₁₈150mm * 4.6mm, 5 μ m; Column temperature: 25 ℃; Detecting wavelength: TP5 is 215nm, and T α 1 is 210nm, and T α 1-TP5 is 210nm; Sample size: 15 μ L; Flow velocity: 0.3 μ L.

Moving phase: TP5 is an acetonitrile: water=20: 80, TFA are 0.1%, and the moving phase of T α 1-TP and T α 1 is ammonium acetate (5mmol/L): acetonitrile=10: 90, trifluoroacetic acid (TFA) is 0.1%.

(2) the above rate of recovery that is drawn of testing: TP5 is 57.97%, and T α's 1 is 70.94%, and T α 1-TP5's is 74.92%.

(3) the external plasma half-life of TP5 and T α 1:

Be respectively 5.6min and 127min the external plasma half-life that records TP5 and T α 1 according to above experiment, and be 140min the external plasma half-life of T α 1-TP5 fusogenic peptide.

2.T α 1-TP5 fusogenic peptide promotes lymphocyte proliferation activity research

Carry out the proliferation experiment of thymosin alpha 1-thymopentin fusion peptide (T α 1-TP5) to splenocyte with conventional mtt assay, the result: T α 1-TP5 fusogenic peptide has significant promotion kunming mice splenocyte proliferation activity.

Above-mentioned activity research confirms: the T α 1-TP5 fusogenic peptide that the present invention makes is compared with TP5, has not only kept its immunocompetence, but also has long half time, active characteristic such as good, will have good application prospect clinically.

Embodiment

Embodiment 1

(1) structure of expression vector pGAPZ α A-T α 1-TP5

1. the design of fusion gene and amplification design T α 1-TP5 fusion gene, its front end contains the EcoRI site, and the rear end contains NotI and zymoplasm site.Increase by conventional PCR system, reactant is carried out the 12g/L agarose gel electrophoresis identify amplified material, and reclaim test kit with PCR and reclaim the PCR product.

2. fusion gene and carrier carry out enzyme and cut pGAPZ α A and PCR product through EcoRI/Not I double digestion, and identify with agarose gel electrophoresis and to connect product.

3. the connection product behind the structure purifying of expression vector is configured to expression vector pGAPZ α A/T α 1-TP5 through the T4 dna ligase, the transformed competence colibacillus intestinal bacteria, and the picking positive colony is identified recombinant plasmid with DNA electrophoresis and sequential analysis.

(2) contain the structure of expression vector pGAPZ α A/T α 1-TP5 engineering bacteria

1. transform expression vector pGAPZ α A-T α 1-TP5 after the Bln enzyme is cut, transform pichia spp GS115 competent cell, make up the engineering bacteria that contains expression vector pGAPZ α A/T α 1-TP5.

2. screening is 10001 μ gmL with containing concentration ^-1Microbiotic Zeocin screens positive colony; Adopt PCR method to identify the fusion gene T α 1-TP5 of positive colony, identify purpose T α 1-TP5 fusogenic peptide with electrophoresis, the aminoacid sequence of fusogenic peptide is shown in SEQ ID NO.1.

(3) expression of T α 1-TP5 fusogenic peptide with separate and identify

1. ferment the pichia spp GS115-pGAPZ α A-T α 1-TP5 engineering bacteria that will make up under 30 ℃ of conditions, fermentation culture is 4～5 days in the PYD substratum.

2. the nutrient solution nickel ion affinity chromatography separation T α 1-TP5 fusogenic peptide of yeast cell is removed in the separation of fusogenic peptide, and identifies with electrophoresis; T α 1-TP5 fusogenic peptide further separates the T α 1-TP5 fusogenic peptide that obtains purifying with Sephadex G-50 after the zymoplasm enzyme is cut.

Wherein:

Described nickel ion affinity chromatography adopts nickel ion affinity chromatography system, comprising:

Binding buffer liquid: 20mmol/LNa ₂HPO ₄, 0.5mol/LNaCl, 20mmol/L imidazoles, pH7.4;

Elution buffer: 20mmol/LNa ₂HPO ₄, 0.5mol/LNaCl, 0.5mol/L imidazoles, pH7.4;

The desalination system of T α 1-TP5 fusogenic peptide: Tris damping fluid, 0.02mol/L Tris alkali, 0.001mol/L EDTA, pH7.4;

Described zymoplasm enzyme is cut and is adopted the zymoplasm enzyme to cut system: 50mmol/LNH ₄HCO ₃Solution, pH8.4,28 ℃ of 4h;

Described SephadexG-50 elutriant is: 50mmol/L (NH4) ₂SO ₄, pH8.0.

Sequence table

＜110〉Shandong University

＜120〉a kind of thymosin alpha 1-thymopentin fusion peptide and preparation method thereof

<141>2008-1-22

<160>1

<210>1

<211>39

<212>PRT

＜213〉artificial sequence

＜221〉thymosin alpha 1-thymopentin fusion peptide

<222>(1)…(39)

<400>1

Glu?Phe?Ser?Asp?Ala?Ala?Val?Asp?Thr?Ser?Ser?Glu?Ile?Thr?Thr?Lys?Asp?Leu?Lys?Glu

1 5 10 15 20

Lys?Lys?Glu?Val?Val?Glu?Glu?Ala?Glu?Gln?Arg?Lys?Asp?Val?Tyr?Leu?Val?Pro?Arg

25 30 35

Claims (2)

1. thymosin alpha 1-thymopentin fusion peptide, it is characterized in that: the aminoacid sequence of described fusogenic peptide is shown in SEQ IDNO.1, and its relative molecular mass is 4470 dalton.

2. the preparation method of the described thymosin alpha 1-thymopentin fusion peptide of claim 1, be made up of following steps:

(1) structure of expression vector pGAPZ α A/T α 1-TP5

Fusion gene T α 1-TP5 increases by the PCR system, and wherein, the leading primer of design contains EcoR I site, and the postorder primer contains Not I and zymoplasm site; Fusion gene T α 1-TP5 behind plasmid pGAPZ α A and the purifying is all used EcoR I and Not I double digestion, agarose gel electrophoresis reclaims carrier and the fusion gene T α 1-TP5 fragment after enzyme is cut, connect 16h with 4 ℃ of T4DNA ligase enzymes and be built into expression vector pGAPZ α A/T α 1-TP5, the transformed competence colibacillus intestinal bacteria, the picking positive colony is identified recombinant plasmid with DNA electrophoresis and sequential analysis;

(2) contain the structure of expression vector pGAPZ α A/T α 1-TP5 engineering bacteria

Expression vector pGAPZ α A/T α 1-TP5 transforms pichia spp GS115 competent cell after the Bln enzyme is cut, make up the engineering bacteria that contains expression vector pGAPZ α A/T α 1-TP5; Positive colony is 1000 μ gmL with containing concentration ^-1The PYD screening of microbiotic Zeocin; Adopt round pcr to identify the T α 1-TP5 fusion gene of positive colony, identify purpose T α 1-TP5 fusogenic peptide with electrophoresis and mass spectroscopy;

(3) expression of T α 1-TP5 fusogenic peptide with separate and identify

Under 30～32 ℃ of conditions, fermentation culture is 4～5 days in the PYD substratum with the pichia spp GS115-pGAPZ α A/T α 1-TP5 engineering bacteria that makes up; Centrifugal removal yeast cell, nutrient solution separates T α 1-TP5 fusogenic peptide with the nickel ion affinity chromatography, and identifies with electrophoresis; Isolating T α 1-TP5 fusogenic peptide further separates the T α 1-TP5 fusogenic peptide that obtains purifying with Sephadex G-50 after the zymoplasm enzyme is cut;

Wherein:

Described nickel ion affinity chromatography adopts nickel ion affinity chromatography system, comprising:

Binding buffer liquid: 20mmol/L Na ₂HPO ₄, 0.5mol/L NaCl, 20m mol/L imidazoles, pH 7.4;

Elution buffer: 20mmol/L Na ₂HPO ₄, 0.5mol/L NaCl, the 0.5mol/L imidazoles, pH 7.4;

The desalination system of T α 1-TP5 fusogenic peptide: Tris damping fluid, 0.02mol/L Tris alkali, 0.001mol/L EDTA, pH7.4;

Described zymoplasm enzyme is cut and is adopted the zymoplasm enzyme to cut system: 50mmol/L NH ₄HCO ₃Solution, pH8.4,28 ℃ of 4h;

Described Sephadex G-50 elutriant is: 50mmol/L (NH4) ₂SO ₄, pH8.0.