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Recombinant factor C from Tachypleus tridentatus expressed by insect cells

A technology of insect cells and factors, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as insurmountable, false positives, batch differences, etc.

Inactive Publication Date: 2013-09-11
SHANGHAI BAISHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are only two kinds of LAL (Limulus amoebocyte lysate) and TAL (Tachypleus Amebocyte Lysate) that can be used in the production of LAL (Limulus amoebocyte lysate) respectively. There are differences in physicochemical properties, such as the optimum pH value for reacting with endotoxin, etc. (6)
[0006]Although the limulus assay (LAL / TAL) has the advantages of high sensitivity (can detect femtogram-level endotoxin) and rapidity, due to the use of complex cell lysis As a reaction raw material, it also has insurmountable defects
For example, the problem of non-specific interference: In addition to reacting with endotoxin, Limulus reagent also reacts with (1-3)-β-D-glucan (1)
Therefore, it is easy to encounter non-specific interference of (1-3)-β-D-glucan in the detection process of Limulus reagent, resulting in false positive results. This is a big test for drugs with complex components, especially traditional Chinese medicine. challenge(7)
Another example is the issue of batch stability: Limulus reagent uses Limulus blood as the raw material for production. Due to differences in seasons and regions, batch differences in Limulus reagent become a common phenomenon.
Moreover, due to environmental pollution, overfishing and other reasons, the population of horseshoe crabs has decreased sharply in recent years and is close to extinction (8), which makes the raw materials for the production of Limulus reagents face the danger of increasing scarcity

Method used

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  • Recombinant factor C from Tachypleus tridentatus expressed by insect cells
  • Recombinant factor C from Tachypleus tridentatus expressed by insect cells
  • Recombinant factor C from Tachypleus tridentatus expressed by insect cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Implementation example 1 Containing the recombinant plasmid pFASTBacHTA-FC of Chinese horseshoe crab C factor to transform DH10Bac competent cells:

[0040] Take 2 μl of pFASTBacHTA-FC plasmid with a concentration of 0.1 μg / μl to transform 100 μl Escherichia coli DH10Bac competent cells, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, quickly place on ice for 5 minutes, add 1ml LB, recover at 37°C for 3.5 hours, and then apply Triple resistance LB plate. After culturing at 37°C for 40 hrs, the color of the colonies was observed.

[0041] The three-resistance LB plate contains 50 μg / ml Kanamycin, 7 μg / ml Gentamicin and 10 μg / ml Tetracycline, 100 μg / ml Bluo-gal and 40 μg / ml IPTG, and the plate is stored at 4°C in the dark.

Embodiment 2

[0042] Implementation example 2 contains the screening and identification of the recombinant bacmid (FC-Bacmid) of Chinese horseshoe crab C factor:

[0043] Pick the white single colony as described in Example 1, re-stretch the LB plate culture, and streak two to three times continuously until no blue colonies are produced. Pick the colony that is still white after streaking several times in a row, put it in the LB test tube containing the third resistance and incubate it at 37°C for 4 hours, and use the bacterial liquid as a template to identify positive clones by PCR.

[0044] PCR system: F: 100pmol, R: 100pmol, dNTPs: 50pmol each, 5×Prime star DNA polymerase buffer: 10μl, Prime star DNA polymerase: 5U, make up 50μl with sterile water.

[0045] PCR reaction conditions: mix the above PCR reaction system, the first stage is 95°C for 3 minutes; the second stage is 95°C, 1min; 54°C, 1min; 72°C, 3min20s; a total of 30 cycles; the third stage 72°C Extend for 10 minutes.

[0046]...

Embodiment 3

[0051] Implementation example 3 contains the extraction of the recombinant bacmid DNA (FC-Bacmid) of Chinese horseshoe crab C factor gene:

[0052] Pick a single colony that was identified as positive by PCR and transfer it to a 5ml LB medium test tube containing 50μg / ml Kanamycin, 7μg / ml Gentamicin and 10μg / ml Tetracycline, shake and culture at 37°C for 24 hours; take 1.5ml of the bacterial liquid, centrifuge at 12000rpm at room temperature 2min, remove the supernatant; gently resuspend the cells with 300μl solution I (15mM Tris-HCl, pH 8.0, 10mM EDTA, 100μg / ml RNase A), then add 300μl solution II (0.2N NaOH, 1% SDS), and rotate gently Mix well and let stand at room temperature for 5 min; slowly add 300 μl of 3M potassium acetate (pH 5.5), mix gently while adding, then place on ice for 5-10 min; centrifuge at 12000 rpm for 10 min, carefully transfer 800 μl supernatant to a Add 800 μl of isopropanol to the EP tube, mix gently, and then put it on ice for 5-10 minutes; 12000 rpm...

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Abstract

The invention relates to the field of bioengineering, in particular to a method for recombining and expressing a gene of a factor C from Tachypleus tridentatus in insect cells so as to detect and remove endotoxin. The method comprises the steps of preparing a recombinant rhabdovirus of the gene of the factor C from Tachypleus tridentatus, transfecting the insect cells, detecting the expression of the recombinant factor C from Tachypleus tridentatus in the insect cells, and the like. The recombinant factor C from Tachypleus tridentatus, prepared by the method, provides a precondition for researching the binding activity of endotoxin of the factor C, and also lays a foundation for the application of the factor C in detection and removal of the endotoxin.

Description

Technical field: [0001] The invention relates to the field of bioengineering, and specifically prepares a recombinant baculovirus containing the Chinese horseshoe crab C factor gene, and uses it to transfect insect cells to express the recombinant Chinese horseshoe crab C factor. Background technique: [0002] Among clinical infusion reactions, pyrogen reaction is the most harmful and has the highest incidence rate. Endotoxin (lipopolysaccharide, LPS) is the most well-studied and most common biological pyrogen, and it is also a problem that major pharmaceutical companies must face, because even pg Trace levels of LPS can also cause severe pyrogen reactions in patients. Therefore, a sensitive and reliable endotoxin diagnostic technique is very necessary. [0003] The amoeba-like cells (amoebocytes) of sea crab have super sensitivity to LPS. When Gram-negative bacteria invade, it can produce a cascade agglutination reaction to the foreign invading bacteria through a series o...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N7/01C12N9/64
Inventor 牟宗春高雪超郭恒昌庄家麟周康
Owner SHANGHAI BAISHENG BIOTECH CO LTD
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