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Expression of human interleukin-12 in craze noctuid and its purifying process

A technology for interleukin and Spodoptera frugiperda, which is applied in the field of human interleukin-12 purification, and can solve problems such as toxicity, immune response, and the amount of hIL-12.

Inactive Publication Date: 2003-05-14
WUHAN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0005] Although studies have shown that hIL-12 has great application prospects and there are many methods for preparing hIL-12, there is a problem with the use of hIL-12. Excessive use will cause the body's immune response and produce toxic effects
Therefore, the use of genetic engineering to obtain hIL-12 for clinical treatment, timely and appropriate provision of patients, is superior to gene therapy, and there is a problem with the safety of introduced viruses in gene therapy
In addition, although some scholars express P35 and P40 subunits separately, the P35 and P40 subunits in hIL-12 are only active through the formation of dimers.

Method used

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Embodiment Construction

[0015] Expression and purification of hIL-12

[0016] (1) Preparation of hIL-12mAb Sepharose CL-4B

[0017] Modified according to the method of March et al. (1974, Anal. Biolchem. 60:149-152).

[0018] with 400ml ddH 2 O wash 100ml Sepharose CL-4B, mix 100ml Sepharose CL-4B and 300ml 2mol / L Na 2 CO 3 Mix and stir. Add 5ml of acetonitrile to 10g of cyanogen bromide, dissolve, then transfer the cyanogen bromide solution to Sepharose CL-4B. Transfer the mixture to a funnel, quickly and gently dissolve with 400ml 0.1mol / L NaHCO 3 (pH9.5), 400ml ddH 2 O and 400ml 0.2mol / L NaHCO 3 (pH 8.5) to wash the mixture. Dissolve 150mg hIL-12mAb in 100ml 0.2mol / L NaHCO 3 (pH8.5). Transfer the Sepharose CL-4B activated by cyanogen bromide to the bottle containing the hIL-12mAb solution, mix at 4°C for 20h, collect the hIL-12mAb-Sepharose CL-4B gel by filtration, wash with 160ml 1mol / L hydrochloric acid, ethanolamine (pH8.0 ) suspension, mixed for 6h. Use 800ml 0.1mol / L NaOAc(pH4.0)-...

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Abstract

The present invention features that recombinant baculovirus AcNPV-hIL12 is hemocele injected to infect craze noctuid larva and after 96 hr the craze noctuid larva is sterilized with 75% concentrationalcohol and its haemolymph is collected via pricking its head with sterilized needle and stored in sterilized tube. The haemolymph is then centrifuged at 100,000 g for 30 min and the supernatant is collected. Human interleukin-12mAb is coupled to Sepharose CL-4B, and hIL-12 is purified with hIL-12mAb Sepharose CL-4B immunoaffinity column. The method of the present invention is convenient, and theobtained interleukin-12 is suitable for treatment of tumor, and bacteria, virus and parasite infected diseases.

Description

technical field [0001] The present invention relates to the expression of human interleukin-12 in Autographa larvae. Specifically, the present invention relates to the efficient expression of recombinant insect virus strains containing human interleukin-12 gene in the living body of Autographa larvae. The human interleukin-12 is expressed, and the invention also relates to a method for purifying the human interleukin-12. Background technique [0002] Human interleukin-12 (human lnterlukin12, hIL-12) is an important cytokine in the human body, also known as natural killer cell stimulating factor (NKSF) or cytotoxic lymphoid maturation factor (CLMF). In 1989, Kobayashi et al. first isolated and purified hIL-12 (Kobayashi M, et al. J Exp Med, 1989ml70:827-845). In 1991, Gubler and Wolf cloned the full sequence of hIL-12 cDNA and expressed it in mammalian cells (Gubler U, et al, Proc Nati Acad Sci USA, 1991, 88:4143-4147; Wolf S F, et al, J Immunol, 1991, 146:3047-3081). hIL-...

Claims

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Application Information

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IPC IPC(8): C07K14/54C12N15/85
Inventor 孟小林徐进平王健鲁伟
Owner WUHAN UNIV
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