Immunological adjuvant with immunity vegulating agent for treating and preventing diabetic from insulin-dependent

An immunomodulator, human insulin technology, applied in DNA recombination technology, protein separation and purification technology and medical related fields, can solve the problems that polypeptide vaccines cannot enhance immunogenicity and cannot be used in human body

Inactive Publication Date: 2006-09-13
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many commonly used adjuvants, such as Freund's complete adjuvant and Freund's incomplete adjuvant, can only be used in animals and cannot be used

Method used

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  • Immunological adjuvant with immunity vegulating agent for treating and preventing diabetic from insulin-dependent
  • Immunological adjuvant with immunity vegulating agent for treating and preventing diabetic from insulin-dependent
  • Immunological adjuvant with immunity vegulating agent for treating and preventing diabetic from insulin-dependent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: Design, synthesis and cloning of HSP65-6×p277 polypeptide gene

[0059] According to the amino acid sequence of MT-HSP65 gene and p277 polypeptide gene, the preferred codons of Escherichia coli were selected, and 6 oligonucleotide fragments were designed with the aid of computer. First, the p277 polypeptide gene was synthesized by PCR method, the gene was cloned into the C-terminus of the L-ansB-C gene of pED, and transformed into Escherichia coli to obtain a recombinant plasmid named pED-p277. Using the pET28a-HSP65 plasmid as a template, the MT-HSP65 gene was obtained by PCR, and the gene was replaced by the L-ansB-C gene in pED-p277, and transformed into Escherichia coli to obtain a recombinant plasmid named pED-HSP65-p277. The p277 gene was cloned repeatedly to obtain the recombinant plasmid pED-HSP65-2×p277; the p277 gene was cloned repeatedly until the recombinant plasmid pED-HSP65-6×p277 was obtained. The specific method is as follows:

[0060] 6 ...

Embodiment 2

[0070] Embodiment 2: Expression of HSP65-6×p277 gene in Escherichia coli

[0071] The recombinant plasmid pED-HSP65-6×p277 was transformed into Escherichia coli BL21. Pick single bacterium colony and inoculate containing 50ug / ml kanamycin LB liquid culture medium from growing different transformant plates, cultivate overnight at 37 DEG C of constant temperature shaking, transfer and inoculate into fresh corn steep liquor liquid culture medium by 1% ratio ( 50ug / ml kanamycin), after culturing at 37°C for 4 hours, adding α-lactose at a final concentration of 0.5mmol / L to induce Escherichia coli to express T7 RNA polymerase, and continue culturing to express the fusion protein HSP65-6×p277. After induction, a small amount of bacterial liquid was taken every 1 hour, and the bacterial cells were recovered by centrifugation. SDS-PAGE electrophoresis and thin-layer scanning showed that the fusion expression of the HSP65-6×p277 polypeptide gene had been realized. The fusion protein r...

Embodiment 3

[0072] Example 3: Separation and purification of recombinant protein HSP65-6×p277

[0073] The engineered bacteria after induced expression were collected by centrifugation, suspended in the cell lysate (pH 8.0, 50mM phosphate buffer, 0.02% lysozyme), stirred at 37°C for 30 minutes, and DNase was added to digest the DNA Until the solution is not viscous, centrifuge to recover the supernatant, and use sulfuric acid to precipitate according to grades. The target protein is mainly in the 35%-45% sulfuric acid precipitate. Redissolve the precipitated fusion protein in pH 6.8 phosphate buffer, dialyze and desalt, and take the supernatant after centrifugation for anion exchange column chromatography, DEAE cellulose DE-52 column (2 × 60cm), using PB / NaCI gradient Elution, collected in sections for SDS-PAGE electrophoresis detection, HSP65-6×p277 in the elution peak at 120-150mmol NaCI, use SDS-PAGE electrophoresis to check the purity of the prepared sample, see Figure 4 . HSP65-1×...

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Abstract

The invention provides the immunomodulator with the free adjuvant having the function of preventing and curing the insulin-dependent diabetes mellitus. Aiming at the weak immunogenicity, the antigen epitope polypeptide gene (a length of antigen epitope p277 rooted from human HSP60) is associated repeatedly six times and inserted repeatedly into the backward position of the heat shock protein HSP65 gene rooted from the mycobacterium bovis, the fusing expression between the repeat series connecting antigen polyeptide and the HSP65 is realized. The produced fusing albumen did not need the adjuvant; the antigen polyeptide don't need be coupled with the carrier chemistry to the immunity. The fusing albumen can inspire the organism to produce the high titer aiming at p277 by means of the mucous membrane immunity, so the nosogeny of NOD chmice diabetic are depressed highly. The invention can prevent the 1 type and 1.5 type diabete characterized in depending on the insulin.

Description

technical field [0001] The invention relates to DNA recombination technology, protein separation and purification technology and medical related fields. More specifically, the present invention relates to the construction, expression and separation and purification of fusion protein HSP65-6×p277. In the field of medicine, the invention is an immunomodulator, which can be used for preventing and treating human insulin-dependent diabetes. Background technique [0002] Human insulin-dependent diabetes mellitus (insulin-dependent diabetes mellitus, IDDM), including type 1 diabetes and type 1.5 diabetes. Type 1 diabetes is an autoimmune disease with familial inheritance, and the majority of patients are teenagers. The main manifestations are the destruction of most of the islet β cells and the absolute lack of insulin. At present, there is no effective treatment, and patients need to rely on insulin for life, which is very painful. After 1992, according to the WHO Diamond Pro...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12P21/02A61K38/16A61P3/10A61P37/02G01N33/53
Inventor 刘景晶金亮王宇朱爱华陈庆梅李建平熊祺琰曹荣月吴洁
Owner CHINA PHARM UNIV
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