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Fusion protein of immunoglobulin fc and human apolipoprotein(a) kringle fragment

A technology of human immunoglobulin and fusion protein, which is applied in the field of fusion protein of immunoglobulin Fc and human apolipoprotein (a) Kringle fragment, which can solve the problems of prolonging half-life and reducing INF-α activity

Inactive Publication Date: 2010-03-03
MOGAM BIOTECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the fusion protein of INF-α and Fc has a very prolonged half-life, it has the disadvantage that the activity of INF-α is reduced (US 5,723,125)

Method used

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  • Fusion protein of immunoglobulin fc and human apolipoprotein(a) kringle fragment
  • Fusion protein of immunoglobulin fc and human apolipoprotein(a) kringle fragment
  • Fusion protein of immunoglobulin fc and human apolipoprotein(a) kringle fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of recombinant vector expressing LK8-Fc fusion protein

[0051] To construct a vector encoding a fusion protein of LK8 and Fc, the LK8 gene (SEQ ID NO: 1) was obtained by PCR using the pET11B vector (WO2001 / 019868) containing the LK8 gene previously prepared by the inventors of the present invention as a template. In addition, the gene (SEQ ID NO: 2) encoding Fc was obtained by PCR using the pRC13-Hpa vector (Korean Patent 467706) as a template. The primers used in each PCR are shown in Table 1 below.

[0052] Specifically, PCR reactions were performed under the following conditions: template DNA was denatured at 94°C for 5 min, followed by 30 cycles of 30 sec at 94°C, 30 sec at 56°C, and 1 min at 72°C, followed by extension at 72°C. 5min. Also, for ease of cloning, a restriction enzyme digestion site was inserted into each primer so that the resulting PCR product had a restriction enzyme digestion site.

[0053]The two gene fragments genera...

Embodiment 2

[0058] Example 2: Establishment of an animal cell line expressing a large amount of LK8-Fc fusion protein

[0059] In order to establish an animal cell line producing the LK8-Fc fusion protein, the pMSG / LK8-Fc constructed in Example 1 was combined with the dihydrofolate reductase DHFR (dihydrofolate reductase) gene (Columbia University, USA) using Dosper (Roche, Switzerland). ) were transfected together into the cell line CHO DG44 (Columbia university, USA) in which the DHFR gene was deleted. Then, by this cell line, the bacterium colonies suitable for the MEM-α minimal medium containing 10% serum were initially selected, and by gradually increasing MTX (Methotrexate; ChoongWaePharma Corporation, Korea) concentration (including 50nM and 1μM) to Selected colonies were subcultured. During secondary culture, among the colonies showing resistance to MTX, a second selection was made for cell lines secreting high amounts of the protein of interest. The selected cell line was cul...

Embodiment 3

[0060] Embodiment 3: Purification of LK8-Fc fusion protein

[0061] In order to purify the LK8-Fc fusion protein, the CHO / LK8-Fc cell line was rotationally cultured in the same manner as in Example 2 in the HyQ-SFM-CHO medium. Such as figure 2 shown. The cells were cultured and the growth and development ability of the cells were observed on the sixth day of culture, and the supernatant was collected by centrifugation. Then, the LK8-Fc fusion protein contained in the supernatant was purified in the following manner. On the basis of the fact that the Fc region of the LK8-Fc fusion protein has an affinity for protein G sepharose (Amersham Pharmacia, USA), affinity column chromatography was performed. Specifically, in a binding buffer (pH 6-8) containing 20-100 mM sodium phosphate, the LK8-Fc fusion protein contained in the supernatant was attached to a protein G sepharose column, and then used glycine buffer (pH 2-5) to elute it from the column ( image 3 ).

[0062] Th...

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Abstract

The present invention relates to an LK8-Fc fusion protein, which has increased angiogenesis inhibitory activity and in vivo stability. More specifically, relates to an LK8-Fc fusion protein in which an LK8 protein having angiogenesis inhibitory activity is fused with the Fc region of human immunoglobulin IgGl, as well as a composition for treating cancer, which contains the fusion protein. The LK8-Fc fusion protein has not only angiogenesis inhibitory activity leading to anticancer and metastasis inhibitory activitivies, but also a very long in vivo half-life, and thus can be used as a more efficient and economic cancer therapeutic agent or cancer inhibitor.

Description

technical field [0001] The present invention relates to the LK8-Fc fusion protein, which can increase the activity of inhibiting angiogenesis (angiogenesis) and the stability in vivo. More specifically, the present invention relates to the Fc region of the LK8 protein which has the activity of inhibiting angiogenesis and human immunoglobulin IgG1 A fused LK8-Fc fusion protein, and a composition for treating cancer, the composition comprising the fusion protein. Background technique [0002] Angiogenesis refers to the process by which new blood vessels are formed from existing blood vessels. It is known that under normal physiological conditions, vascular epithelial cells remain in a difficult to proliferate state, and that angiogenesis occurs only under extremely limited circumstances, including the female menstrual cycle. Dysregulation of the mechanisms controlling angiogenesis can lead to many pathological diseases, including cancer, diabetes, retinopathy, rheumatoid arth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
CPCC07K2319/31C07K14/775A61P17/06A61P19/02A61P27/02A61P29/00A61P35/00A61P35/04A61P43/00C07K19/00
Inventor 柳贤京尹烨安珍衡任仁焕李虎政金长盛朴斗鸿
Owner MOGAM BIOTECH RES INST
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