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Active factor for enhancing lymphocyte targeting killing tumors and preparation method thereof

A technology for lymphocytes and active factors, which is applied in the field of construction and preparation of new biological active factor fusion proteins, and can solve problems such as difficulties in technical means

Inactive Publication Date: 2012-09-26
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For tumor cells to trans-present IL-15, the first method is to transfect tumor cells with IL-15Rα, but how to make tumor cells in vivo specifically take up exogenous gene carrier is still difficult with the current technical means

Method used

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  • Active factor for enhancing lymphocyte targeting killing tumors and preparation method thereof
  • Active factor for enhancing lymphocyte targeting killing tumors and preparation method thereof
  • Active factor for enhancing lymphocyte targeting killing tumors and preparation method thereof

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Experimental program
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Embodiment Construction

[0031] 1. Construction of dsNKg2D-IL-15 gene recombinant expression vector:

[0032] 11 Primer sequences for PCR amplification: Using bioinformatics means, design corresponding primers (see Table 1) to amplify gene fragments of sNKG2D (a), sNKG2D (b) and IL-15. Add the reverse complementary sequence of (Gly4Ser)4 (shown in the box in the table) to the downstream primer sequences of sNKG2D(a) and sNKG2D(b), and take into account the codon degeneracy principle to improve the translation of the protein efficiency. figure 1 The construction strategy of the dsNKg2D-IL-15 fusion gene fragment is shown.

[0033] Table 1. Primer sequences for sNKG2D (a), sNKG2D (b) and IL-15 gene amplification

[0034]

[0035] The PCR amplification product sequence of sNKG2D(a) is shown in SEQ ID NO: 2, wherein the 1st-6th base is the sequence of the BamH I restriction site; the 7th base is randomly added to avoid frameshift mutation of NKG2D nucleotides; 8th-425th is the gene sequence of the N...

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Abstract

The invention relates to an active factor for enhancing lymphocyte targeting killing tumors and a preparation method thereof, wherein the amino acid sequence of the active factor is showed as SEQIDNO: 1. The preparation method comprises the following steps of: respectively amplifying the sections of SEQIDNO:2-4 by utilizing PCR (Polymerase Chain Reaction), inserting the three sections into a PQ31vector in series, and connecting flexible sections among all sections to obtain a fusion protein expression vector; transforming the fusion protein expression vector into an M15 escherichia coli strain, carrying out IPTG inductive expression, then washing an inclusion body, purifying by using Ni+-ITA resins under the condition of denaturation, and further renaturing to obtain a dsNKg2D-IL-15 protein. The preparation method can express a large number of dsNKg2D-il-15 proteins in vitro, and the expressed protein is stable and not degradable in physiological environment, and has the ability of ensuring that tumor cells are in anti-form presentation on IL-15 to activate the activities of NK cells.

Description

technical field [0001] The present invention relates to the construction and preparation of a fusion protein of a novel bioactive factor (dsNKG2D-IL-15, that is, dual soluble NK cell activating receptor NKG2D and cytokine IL-15): based on the use of tumor cell self-binding, enrichment, and The trans-presentation of the active protein not only directly activates NK or cytotoxic T cells (CTL) in its local microenvironment, but also is expected to enhance the specific immune response in the body, which can be used for the treatment of tumors and belongs to the field of biotechnology. Background technique [0002] Tumor cells reduce the expression of HLA class I molecules and co-stimulatory molecules, escape the surveillance of T lymphocytes in the body, but activate the activity of NK cells. If the activating signal transmitted by activating receptors on the surface of NK cells is stronger than the inhibitory signal transmitted by inhibitory receptors, NK cells will be activate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63
Inventor 龚卫娟杨悌潘兴元季明春
Owner YANGZHOU UNIV
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