Tachypleus tridentatus recombinant factor C based on human cell expression and application thereof
By preparing the recombinant factor C of the horseshoe crab in the human cell expression system and constructing a fluorescence reaction system, the problem of false positive and poor stability of traditional horseshoe crab blood reagents in the endotoxin detection was solved, and endotoxin detection with high sensitivity, specificity and stability was achieved.
Patent Information
- Application Number
- CN202510272095.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-06-06
AI Technical Summary
Traditional horseshoe crab blood reagents are susceptible to nonspecific interference in endotoxin detection, resulting in false positive results, and have poor stability, and there are challenges in raw material supply and cost.
Through genetic engineering technology, human cell expression system is used to prepare the recombinant factor of horseshoe crab, and a fluorescence reaction system is constructed to achieve sensitive and accurate detection of endotoxins.
This method can specifically identify and bind endotoxins, improve the sensitivity and accuracy of detection, and has lower preparation cost, no animal ethical disputes, and is suitable for endotoxin detection in various complex samples.
Smart Images
Figure CN120098101A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of genetic engineering, and in particular to a horseshoe crab recombinant factor C expressed based on human cells and an application thereof. Background Art
[0002] The production of traditional horseshoe crab blood reagents relies on horseshoe crab blood. Even if horseshoe crabs are released after blood collection, their survival and reproductive capacity will be greatly reduced, accelerating the decline in the number of horseshoe crabs. As horseshoe crabs are listed as protected animals, this further poses greater challenges to the production and use of horseshoe crab blood reagents. At the same time, traditional horseshoe crab blood reagents are easily affected by nonspecific interference during the detection of endotoxins, resulting in false positive results, and have poor stability.
[0003] In summary, although traditional horseshoe crab blood reagents have high sensitivity, they have significant disadvantages in terms of nonspecific interference, poor stability, raw material supply, cost and legal restrictions. Summary of the invention
[0004] The object of the present invention is to provide a horseshoe crab recombinant factor C with high sensitivity, high specificity and good stability.
[0005] To achieve the above object, the present invention provides the following technical solutions:
[0006] The present invention proposes a horseshoe crab recombinant factor C expressed based on human cells, and the codons of the horseshoe crab recombinant factor C are shown in sequence ID 1.
[0007] Furthermore, the horseshoe crab recombinant factor C is a recombinant protein prepared based on a protein constructed from human cells.
[0008] The present invention also includes the use of any of the above-mentioned horseshoe crab recombinant factor C in endotoxin detection.
[0009] Furthermore, the object of detection is one or more of medicines, biological products, medical devices, food and drinking water.
[0010] Compared with the prior art, the horseshoe crab recombinant factor C disclosed in the present invention can specifically identify and bind to endotoxins, and by constructing a fluorescent reaction system, sensitive and accurate detection of endotoxins can be achieved. Compared with traditional horseshoe crab blood reagents, the horseshoe crab recombinant factor C used in the present invention has the advantages of controllable preparation, lower cost, and no animal ethics controversy. In addition, the horseshoe crab recombinant factor C prepared by the human cell expression system of the present invention has better stability and is suitable for endotoxin detection in various complex samples.
[0011] Instruction Manual
[0012] Figure 1This is a schematic diagram of the expression of horseshoe crab recombinant factor C in 293T cells after transfection at different times and puromycin resistance screening (green represents the translation expression of the gene expressing horseshoe crab recombinant factor C, and red represents the translation expression of the gene expressing transposase).
[0013] Figure 2 This is a schematic diagram of the expression of horseshoe crab recombinant factor C in MCF-7 cells after transfection at different times and puromycin resistance screening (green represents the gene translation expression of horseshoe crab recombinant factor C, and red represents the gene translation expression of transposase).
[0014] Figure 3 This is a schematic diagram of the results of flow cytometry detection of the expression of horseshoe crab recombinant C factor in 293T and MCF-7 cells.
[0015] Figure 4 This is a schematic diagram of the results of fluorescence quantitative PCR detection of the expression of horseshoe crab recombinant C factor in 293T and MCF-7 cells after one month of puromycin selection.
[0016] Figure 5 This is a schematic diagram of the results of fluorescence quantitative PCR detection of the expression of horseshoe crab recombinant C factor in 293T and MCF-7 cells after five consecutive preservation / resuscitations.
[0017] Figure 6 This is a schematic diagram of the results of Western blot detection of the expression of horseshoe crab recombinant factor C in 293T and MCF-7 cells (the red box corresponds to the horseshoe crab recombinant factor C).
[0018] Figure 7 Schematic diagram of different types of fluorescent peptide substrates.
[0019] Figure 8 This is a schematic diagram of the fluorescence spectrum data results caused by different concentrations of bacterial endotoxin (0.001-4.0EU / ml). DETAILED DESCRIPTION
[0020] The technical scheme of the present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.
[0021] By genetic engineering technology, human tumor cells were used as an expression system to prepare horseshoe crab recombinant factor C with biological activity (Note: the horseshoe crab recombinant factor C of the present invention is synthesized, not extracted). The codons of the horseshoe crab recombinant factor C are shown in sequence ID 1.
[0022] Example 1
[0023] stability
[0024] (1) The expression systems of human embryonic kidney cell 293T and human breast cancer tumor cell MCF-7 were constructed respectively, and the constructed cells were screened by puromycin. The expression of horseshoe crab recombinant factor C was observed by confocal microscopy after transfection at different times. Figure 1 and Figure 2 As shown, after screening for the puromycin resistance gene, an expression vector system that stably expresses horseshoe crab recombinant factor C (green) was obtained.
[0025] (2) After one month of stable expression culture, the expression of horseshoe crab recombinant C factor was further detected by flow cytometry. Figure 3 As shown, 293T and MCF-7 cells can still stably express horseshoe crab recombinant C factor.
[0026] (3) In order to ensure that the horseshoe crab recombinant factor C can be stably expressed in the expression system, the expression of horseshoe crab recombinant factor C in 293T and MCF-7 cells was further verified by fluorescence quantitative PCR technology. Figure 4 As shown, one month after puromycin resistance selection, the expression level of horseshoe crab recombinant C factor in 293T was 160,000 times that of the control group, and the expression level in MCF-7 was nearly 140,000 times that of the control group.
[0027] (4) In order to further study the effect of repeated seed preservation / resuscitation on the expression of horseshoe crab recombinant C factor in the vector, the expression of horseshoe crab recombinant C factor in 293T and MCF-7 cells was studied after repeated seed preservation / resuscitation for 5 times. Figure 5 As shown, the expression level of horseshoe crab recombinant factor C in 293T was 71,000 times that of the control group, and the expression level in MCF-7 was nearly 75,000 times that of the control group, indicating that the horseshoe crab recombinant factor C can still be stably expressed in 293T and MCF-7 cells after repeated freezing and thawing.
[0028] (5) The expression of horseshoe crab recombinant factor C was further verified by Western blot experiment. Figure 6 As shown, horseshoe crab recombinant factor C was stably expressed in both 293T and MCF-7 cells.
[0029] Example 2
[0030] Specificity and sensitivity validation
[0031] Design multiple fluorescent peptide substrates (see Figure 7 ), through the endotoxin detection sensitivity test, a fluorescent peptide substrate sequence with good specificity and sensitivity was obtained: FITC-Asp-Pro-Arg-Lys (Dabcyl).
[0032] Using FITC-Asp-Pro-Arg-Lys (Dabcyl) as a fluorescent peptide substrate, the detection ability of recombinant horseshoe crab factor C for bacterial endotoxin was investigated: different concentrations of bacterial endotoxin were set between 0 and 4.0 EU / ml and detected under the same experimental conditions.
[0033] from Figure 8 It can be seen that when the concentration of bacterial endotoxin added is 0, the fluorescence intensity is very weak and almost zero, indicating that the horseshoe crab recombinant factor C has excellent specificity in detecting endotoxin. When the fluorescence increases from 0 to 4.0EU / ml, its fluorescence spectrum shows an increasing trend. At the same time, endotoxins as low as 0.001EU / ml can still be detected, indicating high sensitivity.
[0034] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the scope of the patent application of the present invention should be included in the scope of protection of the claims of the present invention.
Claims
1. A horseshoe crab recombinant factor C expressed based on human cells, characterized in that: The codons of the horseshoe crab recombinant factor C are shown in sequence ID 1.
2. The horseshoe crab recombinant factor C expressed by human cells according to claim 1, characterized in that: The horseshoe crab recombinant factor C is a recombinant protein prepared based on a protein constructed from human cells.
3. The use of horseshoe crab recombinant factor C according to any one of claims 1 to 2, characterized in that: Application in endotoxin detection.
4. The use of horseshoe crab recombinant factor C according to claim 3, characterized in that: The object of detection is one or more of medicines, biological products, medical devices, food and drinking water.