Thymic peptide fusion protein as one new interferon and its prepn. and use

A technology of fusion protein and thymosin, which is applied in the field of DNA recombination technology and medicine, can solve the problems of cumbersome preparation, separation and purification, and high cost of medicines, and achieve the effect of avoiding cumbersome operations, avoiding duplication of labor, and avoiding high-cost operations

Inactive Publication Date: 2005-03-02
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, IFN-α and THY preparations are produced separately, and clinically IFN-α and THY are also administered separately, which leads to high drug costs
In addition, the preparation (especially the separation and purification) of IFN-α and THY is also cumbersome

Method used

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  • Thymic peptide fusion protein as one new interferon and its prepn. and use
  • Thymic peptide fusion protein as one new interferon and its prepn. and use
  • Thymic peptide fusion protein as one new interferon and its prepn. and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Synthesis of IFN-THY fusion gene and construction of expression plasmid in Escherichia coli

[0055] In order to make the IFN-THY fusion protein have the correct spatial conformation, GGGGS was used as the connecting peptide to connect IFN and THY. According to the IFN gene sequence and the nucleotide sequence of the connecting peptide, a pair of primers IFNPRI1 and IFNPRI2 were designed and synthesized, and their nucleotide sequences were:

[0056] IFNPRI1: 5'GCCATATGTG CGATCTGCCT CAAACC 3' (SEQ ID NO: 3)

[0057] IFNPRI2: 5' ATGGATCCAC CACCGCCTTC CTTACTTCTT AAACTTTC 3' (SEQ ID NO: 4)

[0058] Among them, IFNPRI1 is the coding sequence of N-terminal of IFN mature protein; IFNPRI2 is the complementary sequence of IFN C-terminal and connecting peptide coding sequence.

[0059] Using IFNPRI1 and IFNPRI2 as primers and the pSK-IFN plasmid containing IFN gene as a template, PCR amplifies the IFN gene sequence with the connecting peptide nucleotide sequence, and introduces...

Embodiment 2

[0070] Construction of Insect Baculovirus Transfer Plasmid pBacPAK-IT Containing IFN-THY Fusion Gene

[0071] The Escherichia coli expression plasmid pET-IT containing the IFN-THY fusion gene was first digested with NdeI, then filled in with Klenow enzyme, and then digested with XhoI, and the fusion gene fragment of IFN and THY was separated with low melting point agarose, and mixed with The insect baculovirus transfer plasmid pBacPAK-IT containing the IFN-THY fusion gene was constructed by digesting with BamHI and filling in with Klenow enzyme, and then connecting with pBacPAK8 digested with XhoI (Fig. 3). The plasmid sequentially contains the IFN gene, the GGGGS linker peptide coding sequence, and the THY gene in the direction from 5' to 3'.

Embodiment 3

[0073] Obtaining Recombinant Insect Baculovirus Containing IFN-THY Fusion Gene

[0074] Take 5 microliters of insect baculovirus transfer plasmid pBacPAK-IT containing IFN-THY fusion gene and 6 microliters of modified virus BmBacPAK6 linearized by Bsu36I digestion, add 100 microliters of serum-free TC-100 medium and mix well. Add 6 microliters of Lipofectin to 100 microliters of serum-free TC-100 medium and mix well. Mix the two evenly, place at room temperature for 15 minutes, add 800 microliters of serum-free TC-100 medium and mix well. Wash Bm N cells previously cultured in a 35mm culture dish twice with serum-free TC-100 medium, and add the transfer plasmid and Lipofectin mixture dropwise, culture at 27°C for 4-5 days, collect the supernatant for the first round Plaque screening. Take 5 microliters of supernatant to infect Bm N cells in a 35mm culture dish, discard the supernatant after 1 hour and add equal volumes of mixed TC-100 medium and low melting point agarose. P...

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Abstract

The present invention provides a fusion protein of interferon and thymulin (IFN-THY fusion protein), DNA sequence for coding said fusion protein, carrier containing said DNA sequence, host cell containing said carrier, method for preparing said fusion protein by using gene engineering and medicine composition containing said fusion protein. Said invented IFN-THY fusion protein not only possesses activity of TFN, but also possesses the immunostimulation activity of THY, can be used for curing the diseases of viral infection and raising immunological function of human body.

Description

technical field [0001] The invention relates to the fields of DNA recombination technology and medicine. More specifically, the present invention relates to a fusion protein of human alpha interferon 2b (HuIFNα2b, hereinafter referred to as IFN) and human thymosin (hereinafter referred to as THY), a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, and a vector containing the The host cell of the vector, the method for preparing the fusion protein by genetic engineering, and the application of the fusion protein in the treatment of diseases such as viral infection and the enhancement of body immunity. Background technique [0002] Interferon was first discovered in 1957, and Isaaca and Lindenmann called it a cell product capable of inducing resistance to homologous or heterologous viral infection. After more than 40 years of research, the genes, protein structures and their receptor genes and protein structures of the two major categories of in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09A61K38/00A61P31/12A61P37/04C07K14/52C07K14/56C07K14/575C07K19/00C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02
CPCC07K2319/00C07K14/56A61K38/00C07K14/57581A61P31/12A61P37/04
Inventor 吴祥甫杨冠珍何志勇吴峻
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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