Lipopolysaccharide conjugated protein and monoclonal antibody and preparation method and usage

A technology for binding proteins and lipopolysaccharides, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., and can solve the problems of accelerating lipopolysaccharide-binding protein genes, body damage, etc.

Inactive Publication Date: 2007-12-05
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Lipopolysaccharide binding protein can significantly promote the combination of lipopolysaccharide and mCD14. Nanbo et al. found that human lipopolysaccharide binding protein can increase the sensitivity of the body to lipopolysaccharide recognition by 1000 times. In the early stage of acute inflammation reaction, when the serum lipopolysaccharide concentration is relatively high When it is low, there is already a small amount of lipopolysaccharide-binding protein involved, and only in the presence of lipopolysaccharide-binding protein can the body respond to a small dose of lipopolysaccharide or bacteria, thereby enhancing the body's anti-infection effect, but in the middle and late stages of the acute inflammatory response , when the serum lipopolysaccharide-binding protein content rises, lipopolysaccharide-binding protein will lead to excessive activation of monocytes / macrophages, and a large amount of secretion of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF- α) and other cytokines, these factors strongly accelerate the transcription of lipopolysaccharide-binding protein gene and the synthesis of lipopolysaccharide-binding protein polypeptide, which will lead to serious damage to the body, septic shock, disseminated intracapillary coagulation (DIC), multiple Organ failure (MOF) (see Eur J Biochem 1999, 260 (1): 183-191), etc., so how to suppress excessive inflammatory response and prevent the occurrence of disseminated intracapillary coagulation and multiple organ failure has become the current clinical practice. An important research direction in the work

Method used

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  • Lipopolysaccharide conjugated protein and monoclonal antibody and preparation method and usage
  • Lipopolysaccharide conjugated protein and monoclonal antibody and preparation method and usage
  • Lipopolysaccharide conjugated protein and monoclonal antibody and preparation method and usage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1: Preparation and identification of the amino-terminal fragment of human lipopolysaccharide-binding protein:

[0105] (11) Obtain the cDNA sequence encoding human NH-LBP, and construct the expression virus:

[0106] Messenger RNA (mRNA) was extracted from hepatocytes of patients who died of infectious shock, reverse-transcribed into a cDNA library, and the double encoding human NH-LBP was amplified with specific primers 5′-GTCGACTGGAGTGGGAATCTAGGA-3′; 5′-AAGCTTATTCTGTGTTGTAACTGG-3′ Strand DNA sequence, connected with pMD19-T Simple Vector, transferred into competent DH-5a bacteria, after the test was correct, double enzyme digestion with Sal I and Hind III, the target sequence was recovered, purified, and treated with the same enzyme as the vector pFASTBAC was ligated to construct plasmid pFASTBAC-NH-LBP. Transfer pFASTBAC-NH-LBP into DH10BAC bacteria, and construct the shuttle plasmid pBacmid-NH-LBP through gene transposition. After the sequencing is correct,...

Embodiment 2

[0150] Example 2: Preparation of Human Phage Antibody Library

[0151] Mononuclear cells were isolated from the peripheral blood of healthy volunteers, and the DNA sequence was extracted from the monocytes. Seven pairs of primers were used to amplify the variable region sequence (Fd) encoding antibody heavy chain IgVH, and four pairs of primers were used to amplify the sequence encoding The variable region sequence of antibody light chain IgVκ and 5 pairs of primers were used to amplify the variable region sequence of antibody light chain IgVλ, digested with Sac I+Xba I and Xho I+Spe I respectively, and pComb3H phagemid was used as The human phage antibody library Fab was established in XL1-Blue bacteria.

[0152] Figure 4 shows the construction of the human phage antibody library and the results of enzyme digestion identification, in which:

[0153] 1: DNA markers (15000, 10000, 7500, 5000, 2500, 1000, 250bp);

[0154] 2: κbank containing pCOmb3H / SacI+XbaI (cut out the ligh...

Embodiment 3

[0190] Example 3: Preparation of Human Anti-NH-LBP Monoclonal Fab Antibody and dsFv Antibody

[0191] (31) Using NH-LBP as an antigen to screen the Fab of the phage antibody library and identify a single strain:

[0192] Coat the enzyme-linked plate with NH-LBP as an antigen, add the phage antibody library after washing the plate, wash the plate after 2 hours of binding, wash off the bound phage, re-infect XL1-Blue bacteria and amplify the antibody library, and the amplified phage antibody The library was subjected to the next round of screening, with a total of 5 rounds of screening.

[0193] After 5 rounds of screening, the phages carrying anti-NH-LBP antibody were significantly enriched, and the binding ability was increased by 1.65×10 4 times, proving that the screening was successful.

[0194] Each single colony was amplified, induced by Isopropylβ-D-1-thiogalactopyranoside (IPTG), secreted and expressed Fab antibody, labeled with NH-LBP, and Fab was used as a Anti-HRP...

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Abstract

The present invention discloses one kind of lipopolysaccharide bindin and its monoclonal antibody, preparation process and use. Through extracting NH-LBP coding cDNA segment from human liver cell, preparing NH-LBP protein with insect sf21 cell, establishing humanized phage antibody base with pComb3H phagemid and screening with NH-LBP as antigen, anti-NH-LBP humanized monoclonal antibodies, Fab antibody and dsFv antibody are prepared. The Fab antibody and the dsFv antibody can combine specifically with the N end of complete human lipopolysaccharide bindin and inhibit the combination between LBP and LPS competively to lower the LPS sensitivity of body, lower LPS caused excessive inflammatory reaction, promote the degradation of LPS in body and prevent diffused blood coagulation inside capillary vessel and multiple organ failure.

Description

technical field [0001] The present invention relates to genetic recombination engineering, in particular to a lipopolysaccharide-binding protein and its monoclonal antibody, and a preparation method and application of the lipopolysaccharide-binding protein and its monoclonal antibody. Background technique [0002] Lipopolysaccharide binding protein (LBP) is mainly produced by hepatocytes (renal proximal tubule epithelial cells, alveolar wall type II cells can also be synthesized in a small amount) and mediates the binding of endotoxin to target cell receptors. Acute-phase stress response protein, which can significantly promote the combination of lipopolysaccharide (LPS) and CD14 (mCD14) on the surface of monocyte / macrophage membrane to form LPS-LBP-CD14 complex, which passes through the membrane of monocyte / macrophage The Toll-Like Receptor 4 (TLR4)-Myeloid Differentiation Protein 2 (MD-2)-MYD88 recognition complex is transduced, and then transduced into the nucleus by poly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C07K16/18C12N15/86C12N15/09A61K39/395A61P29/00A61P31/04
CPCY02A50/30
Inventor 葛晓冬
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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