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Serine protease inhibitor and preparing method thereof

A technology of serine proteases and inhibitors, which is applied in protease inhibitors, botany equipment and methods, biochemical equipment and methods, etc., and can solve problems such as short half-life

Inactive Publication Date: 2010-06-23
肖懿
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The short half-life is the biggest shortcoming of TFPI when it plays a therapeutic role. It must be administered continuously for 72 to 96 hours to maintain the curative effect. Each course of treatment requires about 160 omg. Such a large dose brings a huge economic burden to the patient

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] (1) Docking and analysis of TFPI and LRP on a computer workstation

[0013] Using InsightH software, TFPI and L were docked on a computer workstation, and the 269, 282, 288 and 289 lysines at the end of the shuttle base of TFP were found to be the key sites for the interaction between the two.

[0014] (2) Design, preparation and characterization of LTFPI

[0015] Lysines at positions 269, 282, 288, and 289 of the TFPI shuttle end were replaced with alanines. The sequence was obtained by PCR point mutation, recombined with pUC19, positive clones were screened by enzymatic hydrolysis, and nucleotide sequence analysis confirmed that the gene sequence was correct. Then the LTFP I gene was cut out and recombined with the prokaryotic expression vector pET28 to form the prokaryotic expression plasmid rLTFP I-pET28. Transform Escherichia coli JM109, extract the plasmid, identify with the corresponding restriction endonuclease, obtain characteristic fragments, and confirm tha...

Embodiment 2

[0017] The method of obtaining the LTFP gene is as follows:

[0018] (1) Using the overlap extension method, select a section of sequence 400bp behind the pET28 multiple cloning site Not1 to design downstream primers: 5'>CCA CTA CGT GAA CCA TCA CCC TAA TCA AGTAAT TAG GCC TCC TTI'TGA'T TCT TTG GAT GAA ACC'Condition, TTT ACATGC CCT CAG ACAAAA GGA GGC CTA ATT Yougu ACC AAA AGA AAA AGA Longgu Youruo CAG AGA GTG AAA ATA GCA TAT<3' Using the plasmid pET28-TFPI as a template, the upstream primer is paired with the overlap extension primer 1, and the downstream primer is matched with the overlap extension primer Extend the primer 2 pair, pCR respectively, recover the product and use it as an intermediate, add upstream primers and downstream primers for PCR, Ncol+Notl double-enzyme digestion of the PCR product, that is, K241A, K254A, K260A, K261A mutant LTFPIO restriction endonuclease Enzyme was purchased from BRL Company, Escherichia coli JM109, plasmid puC19 Invitrogen Company. The c...

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PUM

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Abstract

The invention relates to a serine protease inhibitor and a method for preparing the serine protease inhibitor, in particular to the long-acting recombination of the serine protease inhibitor and the method for preparing the serine protease inhibitor, which belong to the field of biotechnology. The invention provides the serine protease inhibitor which has long half life and good anticoagulation. In the invention, TFPI and LRP molecules are first modeled on a workstation of a computer, the TFPI molecules and the LRP molecules are butted, the half life of the LTFPI is extended by more than ten times compared with the TFPI after replacement and deletion mutation of certain amino acid in 267 to 304 regions at the rear end of TFPI carboxyl in varying degrees, and the LTFPI has the functions of anti-tissue factor, blood coagulation factor VII / Vlla and blood coagulation factor X / Xa. The invention also provides the method for preparing the LTFPI, and the method comprises preparing cDNA of an LTFPI coded sequence part at least containing expressive biological activity, cloning cDAN segments in expressible carriers, using the carriers to transfect host cells, culturing the host cells when the cDNA segments are suitable to be expressed, and recovering and purifying needed LTFPI in cultures.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to long-acting recombination of serine protease inhibitors and a preparation method thereof. Background technique [0002] Serine protease inhibitor (TFPI), the molecular weight is about 40kd, the precursor has 304 amino acids, and the mature protein has 276 amino acids. The latter consists of an amino terminal, three tandem Kunitz-type domains and a spindle terminal. Animals and humans Experiments have confirmed that TFPI can effectively treat severe infection, hypertoxemia, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), hypercoagulable state, thromboembolic diseases and tumors, etc. A protein with good application prospects. Most TFPI is synthesized and secreted by vascular endothelial cells, smooth muscle cells, cardiomyocytes and fibroblasts can also produce TFPI under certain conditions. Whether it is the human body's own TFPI or the recombinant wild-typ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C12N15/15C12N15/63
Inventor 肖懿
Owner 肖懿