Preparation method and application of recombinant canine interleukin-2

An interleukin and recombinant protein technology, which is applied in the field of the preparation of recombinant canine interleukin-2, can solve the problems of little research on recombinant protein and no eukaryotic expression, and achieves high production efficiency and promotes canine lymphocyte proliferation. , the effect of low production cost

Inactive Publication Date: 2014-10-29
NANJING POLICE DOG RES INST OF MINIST OF PUBLIC SECURITY
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Problems solved by technology

[0003] In 1995, Knapp DW et al first reported the cloning of CaIL-2 cDNA, and related research has entered another stage. However, there have been few studies on recombinant proteins, and most of them are expressed in prokaryotes. to report

Method used

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  • Preparation method and application of recombinant canine interleukin-2
  • Preparation method and application of recombinant canine interleukin-2
  • Preparation method and application of recombinant canine interleukin-2

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Experimental program
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Embodiment Construction

[0044] 1. Amplification of canine interleukin-2 mature protein gene

[0045] (1) Isolation and activation of canine peripheral blood lymphocytes

[0046] Aseptically collect peripheral venous blood from dogs, separate lymphocytes with lymphocyte separation medium, wash with PBS two to three times, centrifuge, and culture in 1640 nutrient solution containing 10% calf serum to make the cell density 5×10 6 / ml, then add ConA to make the final concentration 20 μg / mL, in 5% CO 2 , Cultivate at 37°C for 18 h.

[0047] (2) Extraction of total cellular RNA

[0048]The cells were divided into 1.5 mL centrifuge tubes, and the supernatant was discarded after centrifugation at 3000 g for 5 min. One-step extraction of total RNA from lymphocytes with Trizol kit: Add 200 μL Trizol Reagent and shake vigorously. Stand at room temperature for 5 min. Then add 40 μL of chloroform, and vigorously invert the centrifuge tube to mix. After standing for 5 min, centrifuge at 12000 g for 15 min at...

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Abstract

The invention belongs to the technical field of molecular immunology, and in particular relates to a preparation method and an application of recombinant canine interleukin-2. The preparation method comprises the following steps: amplifying mature protein genes of the canine interleukin-2 from canine peripheral blood lymphocytes via a PCR method and connecting the genes onto a pichia pastoris expression vector pPICZaA so as to construct a recombinant vector of the mature protein genes of the recombinant canine interleukin-2; integrating the recombinant vector into a specific position in pichia pastoris in an electrical transformation manner. Thus, the high-efficiency expression of the canine interleukin-2 in the pichia pastoris is realized, and the expressed recombinant protein is good in biological activity. The method is low in production cost and high in production efficiency, and the recombinant canine interleukin-2 is good in biological activity. Thus, the method is suitable for enterprise production.

Description

1. Technical field [0001] The invention belongs to the technical field of molecular immunology, and specifically relates to a preparation method and application of recombinant canine interleukin-2. 2. Background technology [0002] Interleukin-2 (IL-2) can activate T cells, NK cells, and macrophages, promote B cells to secrete antibodies, induce T cells to secrete cytokines, and can comprehensively improve the body's cellular and humoral immune functions. immune modulators. Therefore, IL-2 can be used as an immune enhancer to improve the immune effect of vaccines. In addition, IL-2 can also be used as an immunotherapeutic agent. Studies have reported that IL-2 can be used in animal immunotherapy and has a good therapeutic effect on tumors. In 1983, Taniguchi et al. successfully cloned the human IL-2 gene. Since then, more than 30 kinds of animal IL-2 genes have been successfully cloned, and IL-2 gene recombinant protein has been obtained. Compared with other animals, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/26C07K14/55A61K38/20A61P35/00A61P37/02
Inventor 朱骞宋珍华秦海斌李刚温海贺星亮张汇东
Owner NANJING POLICE DOG RES INST OF MINIST OF PUBLIC SECURITY
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