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Recombinant human alpha-prothymosin interleukin 2 gene and its expression and use

A technology of prothymosin and interleukin, applied in gene therapy, recombinant DNA technology, peptide/protein components, etc., can solve the problems of high toxicity and side effects, achieve high biological activity, and overcome safety problems

Inactive Publication Date: 2003-08-13
广州白云山拜迪生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when IL-2 is used in large doses in the body, the toxic and side effects are relatively large

Method used

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  • Recombinant human alpha-prothymosin interleukin 2 gene and its expression and use
  • Recombinant human alpha-prothymosin interleukin 2 gene and its expression and use
  • Recombinant human alpha-prothymosin interleukin 2 gene and its expression and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Thymocyte total RNA extraction and reverse transcription PCR

[0018] Take 3-4 month old fetal thymus, prepare single cells, suspend in RPMI1640 medium, cell density is 1×10 7 ml, under the combined stimulation of 20ug / ml PHA and 500U / ml IL-2 at 37°C, 5% CO 2 Cultivate under the conditions for 24 hours, take the cultured thymocytes and extract total RNA according to the method of TAKARA total RNA kit, dissolve in DEPC-treated deionized water, take a certain amount of RNA, add downstream primer (P2) and dNTP, in Reverse transcriptase was used for 30 minutes at 42°C to reverse transcribe α-prothymosin mRNA into single-stranded cDNA, and further add upstream primer (P1) and Taq enzyme for PCR to obtain α-prothymosin cDNA. The upstream and downstream primers are as follows: upstream primer 5'GTC AAG CTT ATG TCA GAC GCA GCC GTA G3'downstream primer 5'ACT GGA TCC TTA GTC ATC CTC GTC GGT CTT C3'

Embodiment 2

[0019] Example 2 IL-2 gene RT-PCR process

[0020] Take 5ml of heparin anticoagulant blood, use the density gradient method to separate peripheral blood PBMCs in the lymphocyte separation medium, and use RPMI1640 to adjust the number of cells to (2~3)×10 6 / ml, add PHA10ug, interleukin-250IU respectively, at 37°C, 5% CO 2 Culture under conditions for 24 to 48 hours; collect the above cells, centrifuge at 1000r / min for 5 minutes, take the bottom cells and wash them with PBS buffer, then extract the total RNA of the cells with guanidine phenol chloroform isothiocyanate in one step. The obtained RNA precipitate was dissolved in diethyl pyrocarbonate (DECP) treated water, identified by glyoxal 1% agarose gel electrophoresis (containing 0.5 μg of ethidium bromide), and the total RNA concentration was measured by a UV spectrophotometer. The OD260nm / 280nm is between 1.8 and 2.0, and stored in a refrigerator at -20°C; operate according to the instructions of TAKARA's reverse transcri...

Embodiment 3

[0021] Example 3 Cloning of human α-prothymosin functional region gene

[0022] Use bio-soft, sequence and other biological software to determine the optimal annealing temperature through computer analysis, avoiding the secondary structure of mRNA that may interfere with the initiation of translation; ensure that there is no specific sequence pairing between the two primers, and that there is specific complementarity between the primer templates Pairing to avoid amplification of unnecessary sequences; the 5' of the upstream and downstream primers described in Example 1 were respectively introduced into BamHI and XbaI restriction sites, and artificially synthesized oligonucleotide primers: human α-prothymosin cDNA As a template, the human α-prothymosin gene fragment was obtained after PCR amplification. After the above fragment was treated with BamHI / XbaI restriction endonuclease, the protein was extracted by phenol / chloroform, and the DNA was extracted by ethanol precipitation,...

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Abstract

A recombinant alpha-thymosingen-interleukin 2 gene composed of humanized alpha-thymosingen and interleukin 2 and its expression and application are disclosed. It has stronger bioactivity, and can be used to prepare the medicines for treating viral infection, tumor and immunodeficiency diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant human alpha thymosinogen-interleukin 2 gene and its expression and application. Background technique [0002] Human α-prothymosin (prothymosin-α) is a polypeptide hormone secreted by the thymus, which consists of 110 amino acid residues and is widely distributed in tissues. α-thymosin has two original functions, one is to participate in cell proliferation as a nuclear protein; the other is to have immunostimulatory activity, regulate the state of cellular and humoral immune function, and improve the self-stability of the immune system. The application of human α-prothymosin includes the treatment of hepatitis, AIDS, etc. and as an immune regulator (including anti-aging and tumor adjuvant therapy). At present, the thymosin mixture isolated and extracted from the thymus of pigs and cattle is widely used clinically as an immunopotentiator. The safety issues o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16A61K48/00A61P31/12C07H21/04C07K19/00C12N15/62C12N15/63
Inventor 黄明张瑞玲丘力功
Owner 广州白云山拜迪生物医药有限公司
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