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Anti-I type diabetes fuse protein and preparation thereof

A technology of fusion protein and virus, applied in botany equipment and methods, biochemical equipment and methods, peptide/protein components, etc., to achieve the effect of stabilizing gene expression products, relieving pain and the threat of infection

Inactive Publication Date: 2012-04-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although autoantigens that cause IDDM include insulin glutamic acid decarboxylase, insulin, heat shock protein, carboxypeptidase H and other antigens, only one autoantigen-insulin was orally administered to animals in the experiment, which induced immune tolerance. Produced a protective effect (Science, 1994, 265: 1237)

Method used

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  • Anti-I type diabetes fuse protein and preparation thereof
  • Anti-I type diabetes fuse protein and preparation thereof
  • Anti-I type diabetes fuse protein and preparation thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] (1) Construct the transfer plasmid pBac-CTBINB-SP containing the fusion gene of CTB and human insulin B chain antigen epitope.

[0030] Two primers P1 and P2 of the sequences shown in SEQ ID No: 2 and SEQ ID No: 3 were designed.

[0031] With the pBac-CTBINS plasmid (seeing No. 200310121132.1 patent application) as a template, the sequences shown in SEQ ID No: 2 and SEQ ID No: 3 are used as primers to amplify to obtain a fusion gene CTBINB-SP with a size of about 430bp, which is obtained by PCR The target fragment was digested by BamH I and EcoR I, and connected to the transfer vector pBacPAK8, which was also digested by BamH I and EcoR I, to construct a baculovirus transfer gene containing the fusion gene of CTB and human insulin B chain epitope. Plasmid pBac-CTBINB-SP.

[0032] (2) Obtaining the recombinant silkworm baculovirus BmBacCTBINB-SP containing the fusion gene of CTB and human insulin B chain antigen epitope: the transfer plasmid pBac-CTBINB-SP was co-transf...

experiment example 1

[0035] [Experimental example 1] Construction of transfer plasmid pBac-CTBINB-SP containing fusion gene

[0036] The pBac-CTBINS plasmid (see my patent application (patent number: 200310121132.1)) was used as a template and P1 and P2 were used as primers for amplification. The reaction conditions were pre-denaturation at 94°C for 5 minutes, amplification at 94°C for 1 minute, and 57°C 1 minute, 1 minute at 72°C, react for 30 cycles, and finally incubate at 72°C for 10 minutes to obtain the target fusion gene CTBINB-SP with a size of about 430bp, and the 5' and 3' ends of the gene carry BamH I and EcoR respectively I site. The above-mentioned fusion gene of interest was digested by BamH I and EcoR I, and then connected to pBacPAK8 (CLONTECH Company), which was also digested by BamH I and EcoR I, to construct a baculovirus containing CTB and human insulin B chain epitope genes Transfer plasmid pBac-CTBINB-SP, after enzyme digestion analysis and PCR to identify the correct gene i...

experiment example 2

[0037] [Experimental example 2] Obtaining recombinant baculovirus of CTB and human insulin B chain antigen epitope fusion gene

[0038] Take 5ul insect baculovirus transfer plasmid pBac-CTBINB-SP containing the fusion gene of CTB and human insulin B chain antigen epitope and 6ul wild silkworm nuclear polyhedrosis virus DNA for co-transfection. Take 6ul Lipofectin (GIBCOBRL company) and add 100ul serum-free TC-100 medium and mix well. The BmN cells previously cultured in a 35mm Dish were washed twice with serum-free TC-100 (GIBCOBRL company) medium, and the transfer plasmid and Lipofectin mixture was added dropwise, cultured at 27°C for 4-5 days, and the supernatant was collected for the second stage. One round of plaque screening. Take 5ul of the supernatant to infect the BmN cells in a 35mm Dish, discard the supernatant after 1 hour and add an equal amount of mixed TC-100 medium and low melting point agarose. Pick plaques after 4-5 days, infect BmN cells for 3-4 days, save ...

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Abstract

The invention relates to a fusion protein of anti-I type diabetes, and a preparation method thereof; the invention provides a fusion gene and a fusion protein which is expressed by the fusion gene; the fusion gene is nucleotide sequence which is shown by SEQ ID NO: 1. the invention also constructs an expression vector containing the fusion gene and a recombinant virus containing the expression vector; the fusion protein provided by the invention can be used for preparing the drugs for curing the I type diabetes.

Description

technical field [0001] The invention relates to an anti-type I diabetes fusion protein and a preparation method thereof. Background technique [0002] Diabetes has become one of the largest public health problems in the world today. Diabetes is divided into type I and type II. Type I is insulin-dependent diabetes mellitus (IDDM), which is produced by insulin secretion defects. Type I diabetes accounts for 5-10% of the total number of diabetes. There are at least 1 million type I diabetes patients in the country, and there are also Upward trend. Patients with type I diabetes must use insulin for many years to maintain their lives, which causes great mental and economic pressure on the family and society, and various complications caused by diabetes are the main cause of disability and death, so the type I diabetes Prevention and control is imminent. Type I diabetes (insulin-dependent diabetes mellitus, IDDM) results from defective insulin secretion and is an autoimmune dis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/866C12N7/01C07K19/00A61K39/106A61K38/28A61P3/10
Inventor 金勇丰张耀洲李霁
Owner ZHEJIANG UNIV
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