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Preparation method and application of recombinant human leucocyte interleukin 27

A technology of interleukin and insect cells, which is applied in the field of preparation of recombinant human interleukin 27 and can solve the problems of HBV drug resistance and the like

Inactive Publication Date: 2013-11-27
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the anti-hepatitis B drug α-interferon is currently recognized as the first choice drug for the treatment of chronic hepatitis B, but its curative effect can only make 25% to 40% of cases obtain effective responses; The short-term curative effect of infection has been affirmed, but long-term application can induce drug resistance of HBV

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of recombinant human interleukin 27

[0027] (1) Cloning of human interleukin 27 gene: using the total mRNA extracted from human blood leukocytes as a template, first amplify the total cDNA by reverse transcription RCR, and then use the cDNA as a template to amplify the complete IL with specific primers Two gene fragments of -27 (EBI3 fragment and P28 fragment); among them, the primer P1 (forward primer) used for the EBI3 fragment: 5'-GGAATTCATG

[0028] AGGAAAGGGCCCCCAGC-3', P2 (reverse primer): 5'-CAAGCTTTTAGTGGTGGTGGTGGTG

[0029]GTGGGAACCCTTGCCCAGGCTCATTGTGG-3', introduce EcoRI and HindIII restriction sites, and clone it into the PH promoter of the vector pFASTbac-dual; primer P3 (forward primer) for the P28 fragment: 5'-CCTCGAGATGTTCCCAAGGCCCCCAG-3', P4 (reverse Primer): 5'-GGCATGCTTAGTGGT

[0030] GGTGGTGGTGGTGGGAACCGGGCTGGGGGCTCAATGT-3', XhoI and SphI restriction sites were introduced, and it was cloned under the P10 promoter of the vector...

Embodiment 2

[0035] Example 2 Recombinant human interleukin 27 inhibits the expression of hepatitis B virus HBsAg and HBeAg

[0036] Add the cultured HepG2.2.15 cell recombinant human interleukin 27 to HepG2.2.15 cells, 0.1 μg / well in 24-well plate, set at 37°C, 5% CO 2 After 24 hours of cultivation in the incubator, the expression levels of HBsAg and HBeAg were detected with the hepatitis B virus surface antigen diagnostic kit and hepatitis B virus e antigen diagnostic kit (purchased from Shanghai Kehua Bioengineering Co., Ltd.). The results showed that the HBsAg level decreased by 65.7%, and the HBeAg level decreased by 74.1%.

Embodiment 3

[0037] Example 3 Recombinant human interleukin 27 inhibits the replication of hepatitis B virus

[0038] Add recombinant human interleukin 27 to HepG2.2.15 cells, 0.1 μg / well in 24-well plate, set at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, the hepatitis B virus (HBV) nucleic acid amplification (PCR) fluorescence quantitative detection kit (the detection kit was purchased from Shenzhen Piji Biological Engineering Co., Ltd.) was used to detect the HBV linked to the nucleocapsid. Hepatitis virus genomic DNA, primers P5: 5'-ATCCTGCTGCTATGCCTCATCTT-3' and P6: 5'-ACAGTGGGGAAAGCCCT

[0039] ACGAA-3', probe: 5'-TGGCTAGTTTACTAGTGCCATTTTG-3', reaction conditions: 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, 60°C for 60 seconds for 40 cycles. The results showed that the level of cccDNA decreased by 55.2%.

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Abstract

The invention discloses a preparation method and application of recombinant human leucocyte interleukin 27. The preparation method comprises the following steps: amplifying EBI3 segment and P28 segment of human leucocyte interleukin 27, and cloning onto a vector pFASTbac-dual; recombining the obtained vector transformation Escherichia coli DH10Bac and a Bacmid plasmid into a recombinant transposition plasmid rBacmid, transfecting rBacmid into sf9 insect cell under the mediation of liposome to obtain recombinant baculovirus; infecting the recombinant baculovirus into the sf9 insect cell; and carrying out sf9 insect cell culture, and purifying the supernatant through a nickel affinity chromatography column and a gel chromatography column to obtain the recombinant human leucocyte interleukin 27. The human leucocyte interleukin 27 has the function of resisting hepatitis B virus infection, and can be used for preparing medicines for resisting hepatitis B virus infection. The recombinant human leucocyte interleukin 27 obtained by the method is more approximate to a natural state, and has high bioactivity; and the method has the advantages of low production cost and high yield.

Description

[0001] technical field [0002] The invention relates to the technical fields of molecular biology, virology and medicine, in particular to a preparation method and application of recombinant human interleukin-27. Background technique [0003] Interleukins are a large class of cytokines, which are secreted by activated T cells after the combination of antigen presenting cells (APCs) and T cell receptors (TCRs), and act as immune regulatory factors. It plays an important role in the regulation of immune and inflammatory responses, and has important clinical application value in the prevention, control and treatment of diseases. [0004] Interleukin 27 (IL-27), a new member of the IL-12 family, was discovered and identified in 2002. It is a heterodimer composed of two subunits, Epstein-Barr virus-mediated molecule 3 (EBI3) and p28. IL-27 and IL-12 have some similar biological characteristics, and they both have a higher level of expression on antigen-presenting cells (APC)....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/24C12N7/01C07K14/54A61K38/20A61P31/20
Inventor 邬开朗胡艳民曹艳华张威吴建国金晶
Owner WUHAN UNIV
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