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New-type long-acting thymulin and its application

A thymosin and long-acting technology, which is applied in the new long-acting thymosin and its application field, can solve the problems of reducing the quality of drug treatment, uneven product quality, and unsatisfactory curative effect, and achieve simple and efficient purification steps, not easy to lose, and realize The effect of high-density fermentation

Inactive Publication Date: 2007-11-07
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the domestically produced thymosin is mainly this type of drug [12] , but the thymosin preparation contains less than 1% of the active ingredient Tα1. Not only is the production of the drug limited by the source of raw materials, but the main ingredients are not purified, and some of them have animal antigenicity. In addition, the product quality is uneven due to different manufacturers. There are many side effects, the curative effect is not very satisfactory, and the market urgently needs replacement
[0010] Thymosin has a definite curative effect and is a commonly used and urgently needed drug in clinical practice. Currently, there are unavoidable shortcomings in the marketed products, which reduce the therapeutic quality of the drug to a certain extent.

Method used

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  • New-type long-acting thymulin and its application
  • New-type long-acting thymulin and its application
  • New-type long-acting thymulin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Cloning Construction of HSA cDNA

[0060] For the extracted human liver tissue mRNA, put 100mg of human liver tissue directly into a mortar, add a little liquid nitrogen, and grind quickly. After the tissue becomes soft, add a small amount of liquid nitrogen, and grind again. into a centrifuge tube and let stand at room temperature for 5 min. Centrifuge at 4°C (12000g) for 10min, and take the supernatant. Add 0.2ml of chloroform, close the cap of the centrifuge tube tightly, shake vigorously by hand for 15s, then place in ice bath for 15min, centrifuge at 4°C (12000g) for 15min, and store the RNA in the upper aqueous phase. Transfer the aqueous phase to another clean centrifuge tube, add 0.5ml of isopropanol, mix well, leave at room temperature for 10min to precipitate RNA, and then centrifuge at 4°C (12000g) for 10min. Remove the supernatant, add 1ml of 75% ethanol to wash the precipitate, vortex, and then centrifuge at 4°C (7500g) for 5min to recover the ...

Embodiment 2

[0065] Example 2: Synthesis of Tα1 gene

[0066] According to the known thymosin α1 gene sequence, two oligonucleotide primers were synthesized, and the two primers were used as templates to amplify the Tα1 gene by PCR. And the restriction sites of BspTI and NotI were introduced at the two ends of Tα1 gene respectively.

[0067] Upstream primer sequence: 5'-ATGCCTTAAGTGATGCTGCTGTTGATACTAGTAGTGAAATTACTACTAAGGATTTGAAG-3'

[0068] Downstream primer sequence: 5'-ATTCGCGGCCGCTTAGTTTTTCAGCTTCTTCAACAACTTCCTTCTTTTCCTTCAAATCCT-3'

[0069] The primers were synthesized by Shanghai Yingjun Biological Co., Ltd. Add 3 μl of 20 μmol / L upstream and downstream primers to the 100 μl reaction system, 10 μl of 2 mmol / L dNTP, 10 μl of 10× reaction buffer, 5 U of pfu DNA polymerase, (dNTP, reaction buffer, and pfu DNA polymerase are all from Shanghai Shen Can Gaming Biotechnology Co., Ltd. product). The DNA was amplified with a PCR instrument from Bio-Rad Company. The PCR conditions were denatu...

Embodiment 3

[0070] Example 3: Construction of HSA-Tα1 fusion protein [23]

[0071]In order to make the expressed fusion protein secreted outside the yeast cells, the pPIC9K plasmid was selected as the vector, and the fusion protein gene was inserted into the EcoRI and NotI sites of the MCS of the vector. The products obtained in Example 1 and Example 2 and the pPIC9K plasmid were respectively digested with EcoRI / BspTI, BspTI / NotI and EcoRI / NotI. The purified HSA cDNA fragment, Tα1 gene fragment and linearized pPIC9K plasmid were ligated with T4 DNA ligase at an appropriate molar ratio, and the ligated product was transformed into Escherichia coli DH5α, supplemented with ampicillin (100 μg / μL) On LB agar plate, select positive clones, extract plasmids, and identify them by enzyme digestion. The positive clones obtained are sequenced by Shanghai Yingjun Biological Co., Ltd., so the positive clone E. coli DH5α (pPIC9K / HSA-Tα1, referred to as pPIC9K / H-T) was obtained, and the construction of ...

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Abstract

The present invention relates to one new kind of long-acting thymulin and its preparation and medical use, as well as DNA sequence coding the protein and host cell carrying the DNA sequence. The long-acting thymulin protein includes one first area with at least 85 % sequence homology with serum albumin, one second area connected to the C-end of serum albumin and with at least 85 % sequence homology with thymulin and one third area connected to the N-end of serum albumin and with at least 85 % sequence homology with thymulin. It may have one or several joining peptides. Experiments show that the long-acting thymulin has the bioactivity similar to that of thymulin as well as long half life, and may be used in treating various kinds of immunodeficiency diseases and various kinds of adaptive diseases.

Description

technical field [0001] The invention relates to the preparation and application of a novel long-acting thymosin protein synthesized by genetic engineering technology, including a recombinant vector containing the gene and a host transformed with the vector. Background technique [0002] The thymus is an important immune organ in the body. It plays an important role in the development of the lymphatic system and the maintenance of the normal function of the immune system, especially in anti-infection, anti-tumor, transplantation and autoimmune diseases. As the body ages, the thymus gland will gradually shrink and degenerate, which will lead to a decrease in the body's tolerance to diseases such as tumors, infections, and immunodeficiency. In 1961, after Miller et al. discovered that the thymus was closely dependent on the immune function of antibodies and the development of lymphocytes, the research on the use of thymosin by humans was initiated. 1966Goldstein [1] Firstly, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/47C12N15/62A61K38/16A61P37/00C07K14/765
Inventor 陈建华张新国闫璐颖殷艺伟唐莉
Owner CHINA PHARM UNIV
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