Recombinant molecule of human immunodeficiency virus film molecule gp120 functional domain and human TGF (Transforming Growth Factor)-beta1

A TGF-, virus technology, applied in antiviral agents, recombinant DNA technology, medical preparations containing active ingredients, etc., can solve the problems of non-specificity, high immunogenicity, large gp120 molecule, etc., and achieve easy purification and recovery. , the effect of high purity and high protein expression

Inactive Publication Date: 2012-07-11
THE AFFILIATED HOSPITAL OF XUZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the incidence of aGVHD has been reduced, and the long-term and short-term survival rates of grafts and patients have been improved, there are still disadvantages that cannot be ignor

Method used

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  • Recombinant molecule of human immunodeficiency virus film molecule gp120 functional domain and human TGF (Transforming Growth Factor)-beta1
  • Recombinant molecule of human immunodeficiency virus film molecule gp120 functional domain and human TGF (Transforming Growth Factor)-beta1
  • Recombinant molecule of human immunodeficiency virus film molecule gp120 functional domain and human TGF (Transforming Growth Factor)-beta1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Acquisition of hmTGF-β1 and C2-C4-Linker DNA Fragments

[0038] 1. C2-C4-Linker DNA fragment

[0039] Peripheral blood mononuclear cells were isolated from patients with epidemic HIV-1 infection in China, total RNA was extracted by Trizol (purchased from invitrogen), and cDNA was obtained by reverse transcription with reverse transcriptase superscript III (purchased from invitrogen), guided by cDNA template and specific primers Next, PCR amplifies the DNA fragment in the C2-C4 region. Specific primers (synthesized by Shanghai Yingjun): upstream primer: 5'-CGCAGGATCCTCTGTCAATTTCACGG-3, which introduces an endonuclease BamHI site; downstream primer: 5'-ACGCGTCGACATACATTGCTTTTCCT-3, which introduces an endonuclease SalI site. Reaction conditions: pre-denaturation at 98°C for 3 minutes, 30 cycles at 98°C for 30s, 50°C for 30s, and 72°C for 60s, and finally extension at 72°C for 10 minutes. Gel recovery kit (purchased from Shanghai Jierui) purified and recovered ...

Embodiment 2

[0043] Example 2 Construction and Identification of hmTGF-β1 and C2-C4-Linker Cloning Plasmids

[0044] Both phusion high-fidelity DNA polymerase and pfx Tag DNA polymerase are blunt-ended clones. After adding A to the end of hmTGF-β1 and C2-C4-Linker PCR products using common Tag DNA polymerase, T4 DNA ligase is used to connect to pCR2. 1-T cloning vector, transformed into competent cells DH5α, selected positive monoclonal strains by blue-white screening, placed in LB medium (containing 50mg / ml ampicillin), cultured at 37°C for 16-18h, and extracted a small amount of plasmid for enzyme digestion identification. The constructed plasmids were named pCR2.1-hmTGF-β1 and pCR2.1-C2-C4-Linker respectively. Select the Apal restriction endonuclease site in both the T vector and hmTGF-β1 sequences to identify pCR2.1-hmTGF-β1 by single-enzyme digestion, and identify pCR2.1-C2-C4-Linker by double-enzyme digestion with BamHI and SalI. See the results separately image 3 and Figure 4 ,...

Embodiment 3

[0045] Example 3 Construction and Identification of Prokaryotic Expression Vector of C2-C4-Linker-hmTGF-β1 Recombinant Molecule

[0046] BamHI and SalI double digestion plasmid pCR2.1-C2-C4-Linker, recover the target DNA fragment C2-C4-Linker, SalI and XhoI double digestion plasmid pCR2.1-hmTGF-β1, recover the target DNA fragment hmTGF-β1, The linearized prokaryotic expression vector pET-28a was digested with BamHl and XhoI, and T4 DNA ligase was used to connect C2-C4-Linker and hmTGF-β1 to pET-28a, transform BL21 (DE3) competent cells, and coat LB homologous medium ( Containing 25mg / ml kanamycin) plates were cultured at 37°C for 16h, picked a single colony in LB medium (containing 25mg / ml kanamycin), and cultured for 16-18h on a shaker at 37°C vigorously, a small amount Extract the plasmid and name it pET-C2-C4-L-hmTGF-β1. BamHI digestion identifies the recombinant plasmid pET-C2-C4-L-hmTGF-β1. 1.5% agarose gel electrophoresis shows a band at about 700bp (See Figure 5 ), w...

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Abstract

The invention provides a recombinant molecule of a human immunodeficiency virus film molecule gp120 functional domain and human TGF (Transforming Growth Factor)-beta1 and a preparation method and an application thereof, belonging to the technical field of biology. The molecule is formed by connecting a gp120 C2-C4 region with the human TGF-beta1 through a flexible linker. The C2-C4-linker-hTGD-beta can be used for activating and enhancing the function of a peripheral blood static CD4+CD25+regulatory T cell, can be selectively applied to a CD4+D25-initial T cell, contributes to transforming the CD4+D25-initial T cell into a CD4+D25-regulatory T cell, and is used for increasing CD4+D25-regulatory T cells in peripheral blood; and a new strategy is provided for clinical treatment of autoimmune diseases and transplanting-related diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a gene recombination and expressed Tregization-promoting protein, a preparation method and application thereof. Background technique [0002] In the study of immune tolerance in recent years, CD4+CD25+ regulatory T cells (Treg) have attracted much attention. Treg cells are a kind of immune regulatory cells, and together with other regulatory cells, play a central role in feedback regulation. Many scholars have found that Treg plays an important role in suppressing autoimmune diseases, allergies and inducing transplant tolerance. But not all CD4+CD25+T cells are regulatory T cells, those with positive Foxp3 (forkhead box P3) transcription factor have this function. The proportion of Treg cells in T cells is very low, accounting for about 5% in the thymus and 5-10% in the periphery. Fortunately, current studies have found that CD4+CD25-T cells can be induced to transform into Treg cel...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61K38/16C12N15/62C07K19/00C12N15/63C12N5/10C12N1/19A61P9/00A61P13/12A61P17/00A61P17/06A61P19/02A61P29/00A61P31/18A61P35/00A61P35/02A61P37/02A61P37/06
Inventor 徐开林曾令宇王东洋陈翀曹江桑威张建军齐共建张倩李振宇
Owner THE AFFILIATED HOSPITAL OF XUZHOU MEDICAL COLLEGE
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