Pichiapastoris expression strain for recombinant duck interleukin 2 and construction method and application thereof

A technology for interleukin and Pichia pastoris, which is applied to a recombinant duck interleukin-2 Pichia pastoris expression strain and its construction and application fields, and can solve the problem that post-translational modification cannot be performed, the purification process is complicated, and the biological activity is low. and other problems, to achieve the effect of enhancing immunity, simplifying separation and purification process, and high conversion rate

Inactive Publication Date: 2010-11-24
JIMEI UNIV
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Problems solved by technology

Therefore, the first technical problem to be solved by the present invention is to provide a Pichia pastoris expression strain of recombinant duck interleukin-2, which can carry out post-expression modification (such as glycosylation) of recombinant duck interleukin-2 , to ensure its biological activity, so as to overcome the defect that post-translational modification cannot be carried out in the prior art, so that its biological activity is low; secondly, the second technical problem to be solved by the present invention is to provide a recombinant duck interleukin -2 secreted and expressed in Pichia pastoris, the expressed protein exists in the supernatant in a soluble form to overcome the complex purification process of recombinant duck interleukin-2 expressed in the form of fusion protein or inclusion body in the prior art Defects

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  • Pichiapastoris expression strain for recombinant duck interleukin 2 and construction method and application thereof
  • Pichiapastoris expression strain for recombinant duck interleukin 2 and construction method and application thereof
  • Pichiapastoris expression strain for recombinant duck interleukin 2 and construction method and application thereof

Examples

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Embodiment 1

[0025] This embodiment mainly constructs a Pichia pastoris expression strain of recombinant duck interleukin-2, and the steps are as follows:

[0026] (1) Amplification of duck interleukin-2 gene (DuIL-2):

[0027] Since the homology of the duck IL-2 gene sequence can be as high as 97%, the PCR primers can be directly designed according to the duck IL-2 gene sequence (AF294322, AY173028, AY193713 and AY232490) registered on GenBank for amplification. Mature duck IL-2 (with its own signal peptide removed). The primer sequences are:

[0028] P1: 5'-GCT GAATTC GCACCTCTATCAGAGAAA-3' (the underline is the EcoR I restriction site)

[0029] P2: 5'-TA GCGGCCGC TTATTTTAGCATAGATC-3' (the underline is the Not I restriction site)

[0030] The target gene was amplified by RT-PCR using the total RNA of duck spleen lymphocytes as a template and the above two primers. Reverse transcription system (20 μL): total RNA 5 μL, Oligo (dT) 1 μL, M-MLV 5×Reaction Buffer 4 μL, dNTP Mixture (10m...

Embodiment 2

[0034] This example is an application of a recombinant duck interleukin-2 Pichia expression strain, that is, the secretory expression of recombinant duck interleukin-2 in Pichia pastoris. The recombinant Pichia pastoris GS115 (DuIL -2) Inoculate into 100mL BMGY medium (1% yeast extract, 2% tryptone, 1.34% yeast nitrogen source, 0.00004% biotin, 1% glycerol, 100mmol / L potassium phosphate buffer, pH=6.0), 28~ Shake culture at 30°C for about 22h to OD 600 When it reaches 2-6 hours, centrifuge at room temperature, collect the bacteria and resuspend in 20mL BMMY medium, culture at 28-30°C, 250r / min shaker, and induce the expression of the target protein. Add 100% methanol every 24h to a final mass fraction of 0.5% to maintain the induced expression of the target protein, and induce for 120h. Then the bacterium was centrifuged at 8000r / min for 10min at 4°C, and the culture supernatant was collected for SDS-PAGE detection (such as Figure 5 as shown, Figure 5 It is the SDS-PAGE d...

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Abstract

The invention discloses a Pichiapastoris expression strain for recombinant duck interleukin 2, and a construction method and application thereof. The chromosome of the strain integrates duck interleukin 2 genes. The invention has the characteristics of convenient operation, high conversion rate, simple production process, high product purity, high activity, contribution to simplifying the subsequent purification process and contribution to large scale production.

Description

technical field [0001] The invention relates to a Pichia pastoris expression strain of recombinant duck interleukin-2 and its construction method and application. The invention belongs to the technical field of recombinant microorganism and recombinant protein preparation. Background technique [0002] In recent years, poultry diseases (especially avian influenza) have repeatedly broken out in the world, and my country has not been spared, which has brought serious threats to the national economy and people's health. Therefore, how to improve the immunity of poultry to reduce or It is particularly important to avoid the invasion of such viral diseases. Duck, the most water fowl raised in my country, is an important storage host for avian influenza virus. Duck interleukin-2 (DuIL-2) is a cytokine secreted by lymphocytes and plays an important role in the immune system. Therefore, if DuIL-2 can be efficiently expressed by genetic engineering methods and applied in immune adj...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/26C12N15/81C12P21/00A61K39/39C12R1/84
Inventor 杜翠红曹敏杰刘静文肖安风
Owner JIMEI UNIV
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