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Recombinant limulus three-factor reagent and method for detecting endotoxin with same

A three-factor, endotoxin technology, applied in the field of bioengineering, can solve the problems of the reduction of the number of limulus, the large demand for quantitative endotoxin detection reagents, etc., to achieve a single component, avoid false positive endotoxin detection, and avoid batch instability Effect

Active Publication Date: 2016-08-17
XIAMEN BIOENDO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Limulus reagent can only be produced by collecting Limulus hemolymph. , Implantable biological materials) and other production units are growing rapidly, and the demand for quantitative endotoxin detection reagents is increasing, which makes the number of horseshoe crabs in the ocean gradually decrease

Method used

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  • Recombinant limulus three-factor reagent and method for detecting endotoxin with same
  • Recombinant limulus three-factor reagent and method for detecting endotoxin with same
  • Recombinant limulus three-factor reagent and method for detecting endotoxin with same

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Embodiment approach

[0046] As a preferred embodiment of the present invention, the recombinant Limulus three-factor reagent comprises the following components by weight:

[0047]

[0048] As a preferred embodiment of the present invention, the recombinant Limulus three-factor reagent comprises the following components by weight:

[0049]

[0050] As a preferred embodiment of the present invention, each of the recombinant Limulus factor C, the recombinant Limulus factor B and the recombinant Limulus coagulase factor is obtained by expressing using the insect baculovirus expression system, and the insect baculovirus expresses The system is a eukaryotic system. Using the expression vector of the polyhedrin strong promoter component in the baculovirus structural gene, many eukaryotic target genes can be expressed effectively or even at a high level. The baculovirus currently used in the expression system Not particularly limited, mainly the baculovirus can infect insect cells and thereby expres...

Embodiment 1

[0071] Embodiment 1 Expression of recombinant Limulus three factors of the present invention

[0072] Step 1: Acquisition and amplification of the Limulus three-factor gene;

[0073] Total RNA in Limulus blood cells was extracted and reverse transcribed into cDNA. Primers were designed according to the coding region sequences of Limulus factor C, Limulus factor B and Limulus coagulase factor in GenBank, and the three factor gene fragments were amplified by PCR to obtain the amplified Limulus factor C, Limulus factor C and Limulus coagulase factor respectively Gene fragments of suborders.

[0074] Step 2: Construction and cloning of recombinant three-factor expression vector;

[0075] The amplified limulus factor C, limulus factor B, and limulus coagulase factor target gene fragments were respectively ligated with pTA2Vector at room temperature (15-25°C) for 30 minutes to obtain the first recombinant plasmid, and then each first Recombinant plasmids were transformed into DH5...

Embodiment 2

[0086] The expression of the recombinant Limulus three factors is exactly the same as that in Example 1, the difference is that in the endotoxin detection, the composition of the endotoxin reaction reagent is different, and the component content of the endotoxin reagent is: endotoxin-free water 35 μL, pyrogen-free Tris buffer solution (pH6.5) 25 μL, Tween-20 (0.5‰ concentration) 5 μL, fluorescent substrate (2mM) 8 μL, recombinant factor C 5 μL, recombinant factor B 6 μL, recombinant coagulase factor 6 μL, and the others were the same.

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Abstract

The invention discloses a recombinant limulus three-factor reagent which comprises the following components: endotoxin-free water, a heat-resource-free Tris buffer liquid, Tween-20, a fluorescent substrate, a recombinant limulus factor C, a recombinant limulus factor B and a recombinant limulus proclotting enzyme factor. The invention provides the recombinant limulus three-factor reagent and a method for detecting endotoxin with the same, three recombinant limulus factors (the proclotting enzyme factor, the recombinant limulus factor B and the recombinant limulus factor C) can be expressed by using an insect baculovirus expression system, the reagent can be used for detecting bacterial endotoxin, and by adopting the method, bypass interference of a factor G can be eliminated, so that the possibility of false positive caused by glucan is effectively avoided; moreover, the defects that the number of limulus is limited and different batches of limulus reagents are not stable are avoided, and thus the reagent can be used as a novel endotoxin detection reagent.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a recombinant limulus three-factor reagent and a method for detecting endotoxin. Background technique [0002] Endotoxin (endotoxin) is the main component of the outer wall of Gram-negative bacteria (G-), also known as lipopolysaccharide (Lipopolysaccharide, LPS). Endotoxin is only released when the bacteria die and dissolve or the bacterial cells are destroyed by artificial methods. Endotoxin is an exogenous pyrogen, which can activate neutrophils, etc., to release an endogenous pyrogen , Acting on the thermoregulatory center to cause fever, shock and even death, so endotoxin detection is a necessary procedure in the manufacture of pharmaceuticals and medical equipment in pharmaceutical companies. [0003] Limulus Amebocyte Lysate (LAL / Tachypleus Amebocyte Lysate, TAL) is the gold standard for the detection of endotoxin in the world so far, and has the characteristics of simplicit...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/579
CPCG01N21/6402G01N33/579
Inventor 张春张轶岭祁静刘晓玫
Owner XIAMEN BIOENDO TECH CO LTD
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