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Method for treating septicemia with human neutrophil peptide (HNP) blocking agents

A technology of neutrophils and blocking agents, applied in the direction of antibacterial drugs, anti-animal/human immunoglobulins, antibodies, etc., can solve the problems of reducing the production, function and biological activity of neutrophil HNP, and achieve The effect of enhancing the therapeutic effect

Inactive Publication Date: 2012-07-11
MAANSHAN GUOSHENG BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, there is no medical and pharmaceutical approach to block, reduce and neutralize the production, function and biological activity of neutrophil HNP for the treatment of diseases such as sepsis caused by bacterial infection

Method used

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  • Method for treating septicemia with human neutrophil peptide (HNP) blocking agents
  • Method for treating septicemia with human neutrophil peptide (HNP) blocking agents
  • Method for treating septicemia with human neutrophil peptide (HNP) blocking agents

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Experimental program
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Effect test

Embodiment 1

[0019] In order to efficiently express siRNA, a lentiviral vector system is used, because lentiviral vectors have a wider host range than retroviral vectors. The gene sequence AGGCACTGCTCTCCAAGAT was assembled into a self-developed lentiviral vector. The lentiviral vector and other vector components are co-introduced into human 293T cells in order to replicate and express the lentivirus. After 48 hours, the lentivirus titer reached 1×10 per mL 13 Plaque forming units (pfu).

[0020] In order to test that the above siRNA can effectively inhibit the expression of HNP, bone marrow cells were prepared from rat bone marrow. Two Wistar rats were sacrificed by cervical dislocation. The whole body was sterilized with 75% alcohol, and a total of 4 femurs were taken. All bone marrow cells were washed out with RPMI1640 (pH7.2) in a sterile environment. Pipet 5 times with a pipette, and pass through a No. 4 needle 5 times to make a single-cell suspension. Centrifuge at 1000 rpm for ...

Embodiment 2

[0022]In order to clarify that anti-human HNP-1 antibody can effectively neutralize the activity of extracellular HNP in neutrophils, two experiments were performed. In the first trial, complex HNP (70% HNP-1, 21% HNP-2 and only 5% HNP-3 were extracted from the mixed sputum of 30 patients with chronic bronchopneumonia, with no detectable HNP- 4) [Refer to literature Khine, et al., Blood 107, 2936-2942 (2006)]. 100 μg / ml HNP complex and anti-human HNP-1 antibody (1, 10 and 200 μg / ml) were incubated at room temperature for 30 minutes, and the mixture was used to act on lung cancer A549 cells at 10 μg / ml, and the cells were lysed after 30 minutes. The activity of Akt in cells was detected by immunoenzyme labeling. The results show that anti-human HNP-1 antibody can block HNP-activated Akt enzyme activity in lung cancer A549 cells [see figure 2 】. In the second experiment, 100 μg / ml HNP complexes extracted from human sputum were incubated with anti-human HNP-1 antibodies (1, 1...

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Abstract

The invention discloses a method for treating septicemia with human neutrophil peptide (HNP) blocking agents. The method comprises the following steps of: preparing small interfering RNA (siRNA) and anti-HNP antibody blocking agents and the like, and blocking, neutralizing and reducing the yield, the function and the biological activity of HNP. The siRNA is prepared in vitro through a design and a chemical synthesis method. A HNP-1 polypeptide is produced through a design to be cross-linked with KLH (Keyhole Limpet Hemocyanin) and then to immunize an experimental rabbit, and a rabbit immune globulin is obtained. The same one HNP-1 polypeptide is used for immunizing a mouse, an anti-HNP antibody variable region is prepared, and then a humanized monoclonal anti-HNP-1 antibody is prepared. Both of the siRNA and the anti-HNP-1 antibody can effectively block, neutralize and reduce the yield, the function and the biological activity of HNP, so that the method can be used for treating septicemia and other related diseases caused by bacterial infections.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a method for treating sepsis with a neutrophil antimicrobial peptide blocking agent, including preparing blocking agents such as small molecule interfering RNA (siRNA) and anti-human HNP antibody, blocking, neutralizing and reducing The production, function and biological activity of granulocyte HNP, and the treatment of sepsis and other related diseases caused by bacterial infection. Background technique [0002] It is known that neutrophils play a key role in the occurrence and development of sepsis and multiple organ failure [see literature Brown, et al., Lancet 368, 157-169 (2006)]. Neutrophils contain a large number of polypeptides, enzymes, small molecules and other substances. They play an important role in killing, lysing and destroying bacteria, viruses and fungi that have been phagocytosed into neutrophils. However, this effect can only...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61K39/395C07H21/04C07K16/18A61P31/04
Inventor 张雪晴刘辉宇
Owner MAANSHAN GUOSHENG BIO TECH
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