A kind of genetic engineering horseshoe crab blood g factor and its preparation method and application

A technology of genetic engineering and G factor, which is applied in the field of genetically engineered Limulus blood G factor and its preparation, can solve the problem that the limulus reagent cannot be directly used to detect fungal infection, cannot completely block endotoxin interference, and the demand for detection reagents is increasing, etc. problems, to avoid excessive consumption and dependence, to avoid interference, and to reduce false positives

Inactive Publication Date: 2020-11-03
GUANGDONG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] However, there are two different activation pathways for the coagulation process of Limulus horseshoe crabs, one is the endotoxin-mediated factor C pathway: Factor C (FC) is activated by binding to endotoxin, and then activates factor B, and the activated factor B will coagulate the zymogen (Proclottingenzyme) into coagulation enzyme (clotting enzyme), coagulation enzyme converts coagulation protein into coagulation protein, and the coagulation protein is cross-linked and dehydrated to form a gel; the other is mediated by 1,3-β-D-glucan The factor G (FG) pathway induced by LAL can also cause similar agglutination reactions and produce false positive results. Therefore, LAL reagents cannot be directly used to detect fungal infections
At present, although there is a method to increase the detection specificity by adding anti-lipopolysaccharide substances in the LAL reagent, it cannot completely block the interference of endotoxin on the G test
In addition, Limulus reagent can only be produced by collecting Limulus hemolymph, and the preparation of Limulus reagent relies heavily on Limulus resources. And the rapid growth of production units such as medical devices (such as disposable syringes, implantable biomaterials), and the increasing demand for fungal infection detection reagents have gradually reduced the number of horseshoe crabs in the ocean

Method used

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  • A kind of genetic engineering horseshoe crab blood g factor and its preparation method and application
  • A kind of genetic engineering horseshoe crab blood g factor and its preparation method and application
  • A kind of genetic engineering horseshoe crab blood g factor and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Codon optimization of Limulus blood factor G α subunit gene and β subunit gene

[0051] In this example, through a lot of research, according to the codon preference of Escherichia coli, the codons of the synonymous genes preferred by Escherichia coli are used to replace the original codons of the Limulus gene while keeping the expressed protein sequence unchanged.

[0052] The nucleotide sequence of the α subunit gene after codon optimization is shown in SEQ ID NO: 1, and the original sequence of the α subunit gene before optimization is shown in SEQ ID NO: 3;

[0053] The nucleotide sequence of the β subunit gene after codon optimization is shown in SEQ ID NO: 2, and the original sequence of the β subunit gene before optimization is shown in SEQ ID NO: 4.

Embodiment 2

[0054] Example 2 Preparation method of genetically engineered horseshoe crab blood G factor

[0055] 1. Obtaining recombinant plasmids

[0056] The codon-optimized α subunit gene and β subunit gene obtained in Example 1 were respectively constructed into pET32a-sumo Vector to obtain recombinant plasmids pETsuom-α and pETsuom-β.

[0057] 2. Induced expression of recombinant plasmids

[0058] Transform the successfully constructed recombinant expression plasmid into E.coli BL21(DE3) competent medium, culture it on LB solid culture plate containing ampicillin for 12 h, pick a single colony containing the recombinant plasmid and add 5 mL of LB containing Amp to culture Shake culture overnight, take 5 μL overnight culture bacteria, add 2ml LB medium (containing Amp), and place in a constant temperature shaker at 37°C for vigorous shaking culture. When the A595nm value reached 0.5 (about 3 hours), one tube was added with IPTG to a final concentration of 1 mmol / L to induce expressi...

Embodiment 3

[0063] Example 3 Verification of biological activity of genetically engineered horseshoe crab blood factor G

[0064] (1) method

[0065] Detection of enzyme activity by enzymatic fluorescent substrate method: Dissolve 5.4 mg of Boc-Glu-(OBzl)-Gly-Arg-MCA fluorescent substrate in 760 μL DMSO solution, store at -20°C in the dark, and store the solution at a concentration of 10 mM. Add 5 μL of 1mM fluorescent substrate and 5 μL of 2000pg / mL (1-3)-β- Mix the D-glucan evenly, put it in a water bath at 37°C for 30 minutes, extinguish the fire at 80°C for 5 minutes, and cool it down to room temperature quickly. The production amount of the product MCA was measured with a microplate reader, the excitation wavelength λex=380 nm, and the emission wavelength λem=440 nm.

[0066] (2) Results

[0067] The α and β subunits alone are inactive and cannot be activated by (1-3)-β-D-glucan. After α and β subunits are assembled into factor G holoenzyme structure, (1-3)-β-D-glucan can activa...

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Abstract

The invention discloses a genetic engineering limulus blood G factor as well as a preparation method and an application thereof. According to the invention, nucleotide sequences of [alpha] subgroup and [beta] subgroup genes of the G factor undergo codon optimization, and optimized sequences are shown as SEQ ID NO:1 and SEQ ID NO:2; meanwhile, a prokaryotic expression vector of G factor gene subgroup optimized sequence is successfully constructed, and the prokaryotic expression vector is transformed into escherichia coli for efficient induced expression; expression protein is extracted and purified, and the two subgroups undergo effective fusion; and finally, bio-activity verification is implemented on target protein obtained from fusion. According to the invention, the [alpha] subgroup andthe [beta] subgroup, which achieve efficient expression in the escherichia coli, are successfully obtained, and the two subgroups are re-assembled, so that the G factor, which is relatively high in activity and purity, is obtained; the preparation method is simple in preparation process and low in cost; the G factor has a good biological function, the G factor can be used for producing a limulusreagent for detecting fungal infection and the G factor can reduce false positive; meanwhile, a demand on wild limulus resource can be greatly reduced; and the G factor has a broad application prospect.

Description

technical field [0001] The invention belongs to the field of biomedicine. More specifically, it relates to a genetically engineered horseshoe crab blood G factor and its preparation method and application. Background technique [0002] The incidence of deep fungal infection is increasing year by year, and the mortality rate remains high due to the lack of effective early diagnosis methods. Currently, the methods for detecting fungal infection include blood culture, tissue biopsy, PCR technology and immunological methods. Among them, blood culture and tissue biopsy are not suitable for early diagnosis due to the long time-consuming culture method and low positive detection rate; PCR can only detect known pathogenic fungal infections, and is not suitable for early diagnosis of rare conditional pathogenic fungal infections; and immunology The method is to screen for multiple fungal antigens, which is easy to miss, time-consuming and uneconomical. The severity of deep fungal ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/70C12N1/21G01N21/64G01N33/569C12R1/19
CPCC07K14/43504C12N15/70G01N21/6428G01N21/6486G01N33/56961
Inventor 张海涛伍俊罗辉吴尚
Owner GUANGDONG MEDICAL UNIV
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