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Novel targeted fusion protein with anti-inflammatory action and use thereof

A targeted, single-chain antibody technology, applied in the field of gene products, can solve problems such as infection

Inactive Publication Date: 2009-07-08
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although such systemic complement inhibitors improve the inflammatory response, since the complement system is an important part of the body's immune defense mechanism, systemic complement inhibition can also cause potential side effects such as infection

Method used

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  • Novel targeted fusion protein with anti-inflammatory action and use thereof
  • Novel targeted fusion protein with anti-inflammatory action and use thereof
  • Novel targeted fusion protein with anti-inflammatory action and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Acquisition of anti-P-selectin single chain antibody targeting complement inhibitor ScFv-Crry and construction of its eukaryotic expression vector

[0055] 1. Obtaining anti-P-selectin single chain antibody

[0056] Use the following method to screen anti-P-selectin single-chain antibody, and the specific method includes the following steps:

[0057] 1. Cell culture and identification: the hybridoma cells secreting P-selectin antibody (constructed according to the method in Xu Tinggui, 1982.) (cultivated in complete RPMI1640 containing 15% bovine serum) Medium. Incubator with 5% CO 2 A mixed gas with a humidity of 98%. The specificity and titer of the monoclonal antibody in the culture supernatant were identified by ELISA method, and the antibody titer reached 1:3200 with obvious species specificity.

[0058] 2. Isolation and purification of mRNA: use rapid preparation and purification of mRNA kit (Promega) and refer to the kit instructions from about 2 × ...

Embodiment 2

[0072] Example 2, Screening of high-expression cell lines of anti-P-selectin single-chain antibody targeting complement inhibitor ScFv-Crry and purification of anti-P-selectin single-chain antibody targeting complement inhibitor ScFv-Crry

[0073] 1. Screening of cell lines with high expression of anti-P-selectin single chain antibody targeting complement inhibitor ScFv-Crry

[0074] In order to obtain higher biological protein molecules closer to natural in terms of molecular structure, physical and chemical properties and biological functions, the recombinant eukaryotic expression plasmid pEE14.1 / ScFv-Crry obtained in Example 1 was transfected into Chinese hamsters using liposomes Ovarian cells in CHO. After 24 hours of transfection, the medium was sucked off, and 10 mL of fresh selective medium DMEM+10% FCS+25 μm MSX was added. at 5% CO 2 The mixed gas was cultivated in a 37°C incubator with a humidity of 98%. After 2 weeks, clones of about 1-2 mm appeared, and the clone...

Embodiment 3

[0077] Embodiment 3, in vivo and in vitro biological activity identification and animal experiments

[0078] 1. Complement lysis inhibition experiment

[0079] To measure the inhibitory activity on complement, 60%-80% confluent CHO cells were separated with ethylenediaminetetraacetic acid, washed twice with DMEM, and then resuspended in DMEM to make the final concentration of 10 9 / L. Add 100mL / L rabbit anti-CHO cell membrane antiserum to the cell suspension and react at 4°C for 30min to sensitize the cells. Then the antiserum was discarded, and the cells were resuspended in NHS diluted with DMEM to a final volume of 50 μL or 100 μL. After 60 minutes at 37°C, the cell viability was measured by placenta blue staining and exclusion method (both live and dead cells were counted). The fusion protein ScFv-Crry was diluted with DEME and added to NHS first, and then added to CHO cell suspension. The final concentration was based on the control CHO cells lysed by 100 g / L NHS which...

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Abstract

The invention discloses a target fusion protein with the anti-inflammatory action, in particular an anti-P lectin single chain antibody target complement inhibitor ScFv-Crry and application thereof. The invention aims to provide the anti-P lectin single chain antibody target complement inhibitor ScFv-Crry with the specific targeting property and application of the ScFv-Crry to the preparation of medicines for treating organism inflammatory reactions. The anti-P lectin single chain antibody target complement inhibitor ScFv-Crry is the fusion protein which is obtained through the connection between an amino end connected with an anti-P lectin single chain antibody ScFv and a complement inhibitor Crry(1-319aa). Experiments show that the ScFv-Crry can remarkably improve the efficiency of the cell cracking mediated by an inhibiting complement, be highly aggregated at an immunity damaged position, and obviously inhibit the generation and development of inflammation. Therefore, the ScFv-Crry can be prepared into target P lectin complement inhibitor new genetic engineering target protein medicines aiming at treating inflammatory reactions. The ScFv-Crry has important contributions to the field of pharmacy and bright application prospect.

Description

technical field [0001] The invention belongs to the field of gene products, in particular to an anti-P-selectin single-chain antibody targeting complement inhibitor ScFv-Crry with specific targeting and its application in the preparation of medicines for treating body inflammation. Background technique [0002] Inflammation is the most common and complicated case process in human diseases, which can occur in any part of the body and in any tissue. Inflammatory response is an important pathological process common to many diseases such as tumors, cardiovascular and cerebrovascular diseases, diabetes, autoimmune diseases and trauma. From the perspective of local pathological changes, they all have a lot in common, that is, they are all an overreaction of the body to the damage of inflammatory factors. At present, a large number of data show that the occurrence of inflammation is closely related to the high expression of P-selectin and the activation of complement. [0003] Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11A61K39/395A61K48/00A61P29/00
Inventor 宋宏彬张传福贾雷立周育森刘雪林徐元勇王勇靳连群孙岩松黄留玉
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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