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Horseshoe crab factor b variant

A technology of factor B and amino acid, which is applied in the direction of using a carrier to introduce foreign genetic material, enzymes, and cells modified by introducing foreign genetic material.

Pending Publication Date: 2019-10-22
SEIKAGAKU KOGYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of the documents discloses a variant of factor B having superior enzymatic properties compared with factor B itself

Method used

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  • Horseshoe crab factor b variant
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  • Horseshoe crab factor b variant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0270] Preparation of Murasame-TFB

[0271] The expression vector of Limulus chinensis factor B mutant (Murasame-TFB) was prepared according to the following procedure.

[0272] (1) Production of Murasame-TFB expression vector

[0273]Using the above TFB / pCA7 as a template, an inverse PCR reaction using phosphorylated primers (primer 11 (SEQ ID NO: 32) and primer 12 (SEQ ID NO: 33)) was performed. The inverse PCR reaction was carried out using Tks Gflex DNA polymerase (manufactured by Takara Bio Co., Ltd.) according to the attached manual. Add restriction enzyme (Dpn I) to the above-mentioned PCR reaction solution to decompose the template, perform phenol / chloroform extraction and ethanol precipitation to separate and prepare DNA, and use DNA Ligation Kit to follow the attached instructions. A ligation reaction (self-ligation) is performed.

[0274] After Escherichia coli was transformed using the ligation reaction solution described above, the vector was amplified and pu...

reference example 3

[0279] Measurement of protease activity (specific activity) of TFB

[0280] A solution of 160 nM TFC, 3.2 μM LPS (derived from Salmonella minnesota R595, weight average molecular weight 1,700 Da, manufactured by List Biological Laboratories), 20 mM Tris-HCl (pH 8.0), and 150 mM NaCl was prepared and statically incubated at 37° C. Leave for 20 minutes to activate TFC. Hereinafter, activated TFC is referred to as "α-TFC".

[0281] 20 μL of a solution of 50 nM TFB, 0.2 nM α-TFC, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 100 μg / mL BSA was prepared and allowed to stand at 37° C. for 1 hour. To this solution, 5 µL of 2 mM Boc-Leu-Thr-Arg-MCA (manufactured by Peptide Laboratories) dissolved in 20% DMF was added, and left to stand at 37° C. for 5 minutes. After adding 0.6 M acetic acid (75 µL) to complete the enzyme reaction, the amount of MCA released from the peptide (proportional to the protease activity (total activity) of activated TFB) was measured with a fluorescence detecto...

Embodiment 2

[0283] Determination of protease activity (specific activity) of Murasame-TFB

[0284] Using Murasame-TFB instead of TFB, the same operation as the above was carried out, and the protease activity of Murasame-TFB was measured.

[0285] As a result of the above test, the protease activity (specific activity) of Murasame-TFB was 416.87±20.50 units / μmol.

[0286] The results of and are shown in figure 1 . The results of the above experiments show that Murasame-TFB has 13.4 times higher protease activity (specific activity) than TFB.

[0287] Evaluation of thermal stability of TFB

[0288] Prepare 10 μL of a solution of 100 nM TFB, 20 mM Tris-HCl (pH8.0), 150 mM NaCl, 100 μg / mL BSA at a given temperature (40 °C, 50 °C, 60 °C, 70 °C, 80 °C, or 90 °C) Let stand for 2 minutes. Thereafter, 10 μL of a solution of 0.4 nM α-TFC, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 100 μg / mL BSA was added to this solution, and the mixture was left to stand at 37° C. for 1 hour. Thereafter...

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Abstract

The present invention provides a technique relating to a horseshoe crab factor B variant and, in its turn, provides a means for highly sensitively assaying an endotoxin. A polypeptide having an aminoacid sequence, in which the amino acid residue at the 193-position in the amino acid sequence of horseshoe crab factor B polypeptide is substituted into a cysteine (Cys) residue, is produced. By preparing a Limulus reagent using a combination of the thus obtained polypeptide with horseshoe crab factor C, an endotoxin can be assayed at a high sensitivity.

Description

technical field [0001] The invention relates to a variant of limulus factor B. Background technique [0002] Means for detecting microorganism-derived substances and measuring the degree of microbial contamination are important in hygienic management of medicines and foods, and in the diagnosis of animals including humans. As a means of measuring the degree of microbial contamination, the Limulus test is becoming popular. The Limulus test is a technique to measure the degree of microbial contamination by using endotoxin (LPS) or (1→3)-β-D-glucan as the target substance, and it uses the protease precursor of Limulus to be absorbed by these target substances. Determination of properties of activation. [0003] As a limulus test, a method using a limulus blood cell extract (lysate of limulus amoeba-like cells; hereinafter, simply referred to as "cell lysate") is widely used. This method utilizes when the precursors of serine proteases (factor C, factor B and coagulation zymo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/15C12N1/19C12N1/21C12N5/10C12N9/50C12P21/02C12Q1/37G01N33/579
CPCG01N33/579C12N9/6408C12Y304/21085G01N2400/50C12N15/70C12N15/79C12N9/50C12Q1/37
Inventor 小林雄毅水村光小田俊男川畑俊一郎
Owner SEIKAGAKU KOGYO CO LTD
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