Recombinant Baculovirus for expressing CTB and human insulin fusion protein and use thereof

A technology of silkworm baculovirus and human insulin, applied in the fields of application, virus/bacteriophage, peptide/protein components, etc., can solve the problems of low tolerance effect and low expression level, achieve high activity, high yield, and relieve pain and the effect of infected threats

Inactive Publication Date: 2006-10-04
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the expression of fusion protein is mainly carried out through transgenic plants. The defect is that the ex

Method used

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  • Recombinant Baculovirus for expressing CTB and human insulin fusion protein and use thereof
  • Recombinant Baculovirus for expressing CTB and human insulin fusion protein and use thereof
  • Recombinant Baculovirus for expressing CTB and human insulin fusion protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] [Example 1] Construction of fusion gene of CTB and human insulin

[0025] First, 6 primers were designed according to the published human insulin gene sequence (Science 1980; 208: 57), in which the C peptide between the B chain and the A chain was replaced by a 6 amino acid "mini-C" peptide (RRGSKR) ( Gene 1981; 16:63) where F1, F2, and F3 are forward primers, R1, R2, and R3 are reverse primers (Table 1), a BamHI site is added to the 5' end of F1, and EcoRI is added to the 5' end of R1 Sites (indicated in bold).

[0026]Obtain human insulin by one chain extension reaction and two PCR methods: (1) chain extension: the reaction system contains primers F3 and R3, 5 units of Taq enzyme and other conventional PCR reagents (Shanghai Shenggong Company); the reaction condition is 94 Denaturation at ℃ for 10 minutes, annealing at 55℃ for 5 minutes, let F3 and R3 serve as primers and templates for each other, and extend at 72℃ for 5 minutes, and the product obtained is denoted a...

Embodiment 2

[0028] [Example 2] Construction of the insect baculovirus transfer plasmid of CTB and human insulin fusion gene

[0029] The above-mentioned target fusion gene was digested with BamHI and EcoRI and then connected to pBacPAK8 (CLONTECH Company) which was also digested with BamHI and EcoRI to construct a bacmid transfer plasmid pBac-CTB-INS containing CTB and human insulin gene. After enzyme digestion analysis and PCR identified that the gene was correctly inserted, automatic sequence determination showed that the constructed fusion gene sequence was completely correct, and the fusion gene sequence and its encoded amino acid sequence were shown in SEQ ID NO.1 and SEQ ID NO.2.

Embodiment 3

[0030] [Example 3] Obtaining of recombinant baculovirus of fusion gene of CTB and human insulin

[0031] Take 5ul insect baculovirus transfer plasmid pBac-CTB-INS containing CTB and human insulin fusion gene 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 the supernatant, lyse the cells with NaOH for Southern hybridiza...

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Abstract

The invention relates to the preparation of polypeptides medicament through genetic engineering, which comprises restructuring fusion gene with CTB and human insuline with Bombyx mori Nuclear polyhedrosis Virus, obtaining the recombinant viruses, inoculating the recombinant virus to domestic silkworm larva and aurelia for expression, freeze drying to obtain the oral medication. Animal test has shown that, the medicament has appreciable actions for lowering rat blood sugar and repressing diabetes, thus a novel method for preparing anti-I type diabetes oral medication can be achieved.

Description

technical field [0001] The invention relates to a recombinant insect baculovirus, the preparation of a fusion protein through the recombinant virus and its application in the preparation of oral medicines against type I diabetes. Background technique [0002] Type I diabetes (insulin-dependent diabetes mellitus, IDDM) results from defective insulin secretion and is an autoimmune disease targeting pancreatic beta cells. 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. A protective effect was produced (Science, 1994, 265:1237). This is because oral administration of antigens can induce regulatory T cells to secrete inhibitory cytokines, such as TGF-β, which can inhibit all pathological immune responses induced by various antigens that are not related to each othe...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N7/01C12N15/33C12N15/62C12P19/34C07H21/04C07K19/00A61K38/28A61P3/10
CPCY02A50/30
Inventor 金勇丰张耀洲龚朝晖
Owner ZHEJIANG UNIV
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