Recombinant tachypleus tridentatus C factor based on codon optimization and application of recombinant tachypleus tridentatus C factor in endotoxin detection

Abstract

The invention discloses a recombinant tachypleus tridentatus C factor based on codon optimization and application of the recombinant tachypleus tridentatus C factor in endotoxin detection, and relates to the technical field of bioengineering. The limulus C factor sequence is used as a starting sequence, the limulus C factor is recoded, and the gene sequence of the recombinant limulus C factor is shown as SEQ ID NO: 1; protein corresponding to the sequence is expressed through an insect cell-baculovirus expression system, and the expression quantity is high, that is, the protein expression quantity of the recombinant horseshoe crab factor C is successfully improved through codon optimization. The production cost of the recombinant tachypleus tridentatus C factor endotoxin detection method kit is reduced, and the recombinant tachypleus tridentatus C factor detection method replaces a tachypleus tridentatus reagent detection method to become a mainstream method for endotoxin detection, so that the problem of raw material supply of tachypleus tridentatus reagent due to the fact that tachypleus tridentatus becomes a protective animal is solved; and the existing recombinant horseshoe crab factor C detection method has the problems of high product price and the like.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a recombinant horseshoe crab Factor C based on codon optimization and its application in endotoxin detection. Background Art

[0002] There is a cascade reaction in horseshoe crabs to cope with endotoxin invasion, that is, the rapid blood coagulation reaction of horseshoe crab hemolymph to endotoxin. Specifically: after endotoxin invades the hemolymph, it binds to horseshoe crab Factor C and activates horseshoe crab Factor C, which is the start of the cascade reaction. Subsequently, the activated horseshoe crab Factor C activates Factor B, and the downstream prothrombin is activated by the activated Factor B to become thrombin. Thrombin acts on procoagulin, causing its C peptide to be released, and the A and B peptide chains are connected by disulfide bonds. This process makes the procoagulin gel-like. Based on this, the horseshoe crab reagent detection method was born, which is used for endotoxin detection in industries such as medical, food, and animal husbandry. Especially in the medical industry, if the endotoxin in medical devices used by patients exceeds the standard, it may cause patients to have fever, shock, or even death. With the development of industries such as medical, the demand for horseshoe crab reagents has increased greatly. The raw material of horseshoe crab reagents, horseshoe crab blood, has become a limiting factor in supply. Worse still, problems such as a large amount of horseshoe crab blood extraction and environmental pollution have caused the number of horseshoe crabs to drop sharply, about 2% of the number of horseshoe crabs a decade ago. In 2019, the International Union for Conservation of Nature officially listed the Chinese horseshoe crab as an endangered animal. Due to the great difficulty in artificially breeding horseshoe crabs, the raw material supply problem of horseshoe crab reagents has become a major problem. For this reason, people actively seek alternative endotoxin detection schemes, and the recombinant horseshoe crab Factor C detection method has emerged. Horseshoe crab Factor C is a key link in the cascade reaction triggered by endotoxin invasion in horseshoe crabs. That is, after binding to endotoxin, horseshoe crab Factor C can be activated, and then activate the downstream cascade reaction. Based on this, in the recombinant horseshoe crab Factor C detection method, if there is endotoxin in the sample to be tested, the endotoxin activates the recombinant horseshoe crab Factor C, and then the activated recombinant horseshoe crab Factor C acts on the fluorescent substrate. By beam excitation, the emitted light can be obtained with an enzyme-labeled instrument to quantitatively detect the endotoxin in the sample. Compared with the horseshoe crab reagent detection method, the recombinant horseshoe crab Factor C detection method has higher sensitivity, higher specificity, accuracy, and quantification ability. In addition, the recombinant horseshoe crab Factor C detection method does not rely on animals, has strong consistency between product batches, and excludes the false positive interference of β-1,3-glucan. Since 2012, it has been successively approved by various countries as an alternative method to replace the horseshoe crab reagent detection method.

[0003] At present, most of the recombinant horseshoe crab Factor C in the recombinant horseshoe crab Factor C endotoxin detection kits on the market follow the original gene sequence of horseshoe crab Factor C. Due to its protein molecular weight as high as 123 kDa, the existing protein expression systems show low expression ability when expressing this protein, which means that the production cost of recombinant horseshoe crab Factor C is greatly increased, restricting the implementation and development of the recombinant horseshoe crab Factor C detection method. Therefore, improving the expression level of horseshoe crab Factor C has become an important breakthrough point for reducing product costs. Summary of the Invention

[0004] The object of the present invention is to solve the problem that the price of the recombinant factor C endotoxin detection kit on the current market is too high. By optimizing the codons of factor C, starting from increasing the expression level of factor C, reducing the volume of enzyme solution added to each reaction, reducing the production cost of the product and thus reducing the sales cost of the product, so as to promote the development of the recombinant factor C detection method, solve the supply problem of the original endotoxin detection method - the horseshoe crab reagent detection method, and promote the development of the medical industry.

[0005] Another object of the present invention is to provide a recombinant factor C based on codon optimization;

[0006] Another object of the present invention is to provide a recombinant plasmid containing the recombinant factor C;

[0007] Another object of the present invention is to provide a recombinant baculovirus containing the recombinant factor C;

[0008] Another object of the present invention is to provide an mRNA transcribed from the gene sequence of the recombinant factor C;

[0009] Another object of the present invention is to provide a recombinant factor C protein encoded by the gene sequence of the recombinant factor C;

[0010] Another object of the present invention is to provide a preparation method of the recombinant factor C protein;

[0011] Another object of the present invention is to provide an endotoxin detection reagent containing the recombinant factor C protein;

[0012] Another object of the present invention is to provide an endotoxin detection kit containing the endotoxin detection reagent;

[0013] Another object of the present invention is to provide an endotoxin detection method.

[0014] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0015] A recombinant factor C based on codon optimization, whose gene sequence is shown in SEQ ID NO:1.

[0016] mRNA folding causes the RNA to exist in the form of the lowest possible free energy. And the lower the energy during folding, the more difficult it is to open the secondary structure of the mRNA. The difficult-to-open secondary structure will hinder the movement of ribosomes, thereby affecting the protein yield. Based on this, the present invention designs a set of codon optimization schemes, optimizes the codons of the gene sequence of factor C, and increases the protein expression level.

[0017] Codon optimization scheme: In the present invention, different nucleotide length windows (30, 40, 50, 60 nt) were set, and the original mRNA sequence of horseshoe crab Factor C was scanned with the RNAfold program to obtain the minimum free energy (MEF) of RNA folding at different window lengths. It was found that when scanning with a 50-nucleotide length window, the minimum free energy could reach -20 kcal / mol.

[0018] Based on the horseshoe crab Factor C sequence as the starting sequence, the present invention recoded the horseshoe crab Factor C. The recoding principles are as follows: 1. Remove six rare codons (ctt, cta, cgt, cga, cgg, ggg); 2. For any RNA fragment with a length of 50 nucleotides, the minimum free energy of folding is not less than -12 kcal / mol.

[0019] The present invention also claims protection for a method for obtaining a recombinant horseshoe crab Factor C gene sequence, comprising the following steps:

[0020] Using the horseshoe crab Factor C mRNA sequence as the starting sequence;

[0021] Remove the following rare codons: ctt, cta, cgt, cga, cgg, and ggg;

[0022] Use the RNAfold program to optimize the sequence so that the minimum free energy of any RNA fragment with a length of 50 nucleotides is not less than -12 kcal / mol.

[0023] The present invention also claims protection for a recombinant plasmid containing any one of the above-mentioned recombinant horseshoe crab Factor C.

[0024] The present invention also claims protection for a recombinant baculovirus containing any one of the above-mentioned recombinant horseshoe crab Factor C sequences.

[0025] The present invention also claims protection for an mRNA of recombinant horseshoe crab Factor C, which is transcribed from the gene sequence of any one of the above-mentioned recombinant horseshoe crab Factor C.

[0026] The present invention also claims protection for a recombinant horseshoe crab Factor C protein, which is expressed in cells by any one of the above-mentioned recombinant horseshoe crab Factor C; the cells are insect cells Sf9 or HighFive cells.

[0027] The present invention also claims protection for a method for preparing a recombinant horseshoe crab Factor C protein, comprising the following steps:

[0028] Transfect or infect Sf9 or HighFive cells with the above-mentioned recombinant plasmid or the above-mentioned recombinant baculovirus;

[0029] Culture the Sf9 or HighFive cells;

[0030] Collect the culture medium to obtain recombinant horseshoe crab Factor C protein.

[0031] The present invention also claims protection for an endotoxin detection reagent, comprising the recombinant horseshoe crab Factor C protein described in any one of the above.

[0032] The present invention also claims protection for an endotoxin detection kit, comprising the above endotoxin detection reagent.

[0033] The present invention also claims protection for an endotoxin detection method, which uses any one of the above recombinant horseshoe crab Factor C proteins, the above endotoxin detection reagent, or the above endotoxin detection kit for endotoxin detection.

[0034] Advantages of the present invention:

[0035] By optimizing the codons of recombinant horseshoe crab Factor C, the present invention increases the protein expression level, reduces the production cost of the recombinant horseshoe crab Factor C detection method kit, and realizes the replacement of the horseshoe crab reagent detection method with the recombinant horseshoe crab Factor C detection method as the mainstream method for endotoxin detection, thereby solving the problems of raw material supply caused by the horseshoe crab becoming a protected animal, the inconsistency between product batches, and the high product price of the existing recombinant horseshoe crab Factor C detection method. It meets the market demand and promotes the development of industries such as medical treatment and food. Description of the Drawings

[0036] The drawings are used to provide further explanation of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, and do not constitute a limitation to the present invention. In the drawings:

[0037] Figure 1 It is a result diagram of the RNAfold program scanning the horseshoe crab Factor C RNA sequence;

[0038] Figure 2 It is a result diagram of the RNAfold program scanning the recombinant horseshoe crab Factor C RNA sequence after the optimization of the method in Example 1;

[0039] Figure 3 It is a result diagram of the RNAfold program scanning the recombinant horseshoe crab Factor C RNA sequence after the optimization of the method in Comparative Example 1;

[0040] Figure 4 It is a comparison of the protein expression of horseshoe crab Factor C (FC group), recombinant horseshoe crab Factor C after the optimization of the method in Example 1 (FC opt group), and recombinant horseshoe crab Factor C after the optimization of the method in Comparative Example 1 (FC opt-H group) detected by SDS-PAGE;

[0041] Figure 5Comparison of the endotoxin detection effects among horseshoe crab Factor C (FC group), recombinant horseshoe crab Factor C after optimizing the method of Example 1 (FC opt group), and recombinant horseshoe crab Factor C after optimizing the method of Comparative Example 1 (FC opt-H group). Detailed implementation mode

[0042] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are, unless otherwise specified, reagents and materials that can be obtained from commercial channels.

[0043] Protein expression involves the processes of DNA replication into DNA, DNA transcription into mRNA, and mRNA translation into protein. Among them, the stability of gene sequences, the secondary structure of mRNA, and the translation process are all crucial for the protein expression efficiency. Therefore, the regulation of protein expression mainly includes the following aspects: First, select a suitable codon combination for the protein expression system, avoid rare codons, and increase the use of preferred codons. Second, simplify the secondary structure of mRNA to prevent complex secondary structures from hindering the translation process of ribosomes and thus affecting expression. In addition, there are also optimizing repetitive sequences and adjusting the GC content, etc.

[0044] Currently, there are many codon optimization schemes on the market. However, the processes involved in protein expression are complex and diverse, and there is currently no unified optimal codon optimization scheme, that is, the schemes that are well optimized for other proteins may not be applicable to recombinant horseshoe crab Factor C. Therefore, after analyzing the mRNA sequence of horseshoe crab Factor C, the present invention proposes this optimization scheme.

[0045] After analyzing the mRNA sequence of horseshoe crab Factor C, the present invention found that when the horseshoe crab Factor C uses a 50-nucleotide length scanning window, its free energy can be as low as -20 kcal / mol. The low free energy will cause the secondary structure of mRNA to be difficult to open, thereby hindering the translation of ribosomes and ultimately resulting in a decrease in protein expression. In response to this, the present invention optimized the mRNA sequence to relatively increase its free energy, reduce the difficult-to-open secondary structure of mRNA, and perform codon optimization for horseshoe crab Factor C to increase its protein expression.

[0046] Example 1

[0047] In this example, the original gene sequence of horseshoe crab Factor C was codon-optimized through genetic engineering to obtain a recombinant horseshoe crab Factor C gene sequence. This sequence was expressed through an insect cell-baculovirus expression system to obtain a protein with a high expression level. Finally, the endotoxin sensitivity of the recombinant horseshoe crab Factor C was detected.

[0048] (1) Obtain the recombinant horseshoe crab Factor C gene sequence (FC opt sequence) after codon optimization

[0049] Using the horseshoe crab Factor C mRNA sequence as the starting sequence; first, six rare codons (ctt, cta, cgt, cga, cgg, ggg) were removed, and then different nucleotide length windows (30, 40, 50, 60 nt) were set, and the RNAfold program was used to scan the mRNA sequence of horseshoe crab Factor C. Taking the 50-nucleotide length as an example, the scan found that: near 0 nt - 500 nt, 1000 nt - 1500 nt, 2000 nt - 2500 nt, and 3000 nt, the free energy of the RNA sequence was lower than -15 kcal / mol (as Figure 1 shown), and the low free energy was not conducive to protein expression. Therefore, the RNA sequence was optimized, and the optimized free energy was higher than -10 kcal / mol (as Figure 2 shown). As Figure 2 can be seen, the overall free energy has increased compared to the original mRNA sequence. According to the optimized mRNA sequence, the optimized recombinant horseshoe crab Factor C gene sequence (FC opt sequence) was obtained, as shown in SEQ ID NO:1.

[0050] (2) Construction of recombinant plasmid pQB3a-FC opt

[0051] The vector pQB3a was double digested with restriction enzymes, and the restriction enzyme sites were: Nco I, BspE I; the pQB3a (NcoI, BspEI) was recovered by gel extraction.

[0052] The FC opt sequence was amplified using primers FC opt-F (ctagataaaaaaccgccaccATGgttttagccagttttct) and FC opt-R (tggtgatggtggtgTCCGGAttagataaactggcggatcc), and the FC opt was recovered by gel extraction.

[0053] The gel-extracted pQB3a (NcoI, BspEI) and FC opt were ligated by homologous recombination and transformed into Escherichia coli Mach T1. After overnight culture, colonies were picked into liquid LB containing antibiotics for expansion culture, and plasmids were extracted to obtain the recombinant plasmid pQB3a-FC opt.

[0054] (3) Construction of recombinant baculovirus vAcBacIIIG / rFC opt

[0055] Add 2 μL of the extracted plasmid pQB3a-Fc opt, 2 μL of qBac-ⅢG and 50 μL of sterile water to a 1.5 mL centrifuge tube and mix; add 50 μL of sterile water and 3 μL of transfection reagent to another centrifuge tube; after mixing the two tubes, let stand for 15 min, and then add them to a 12-well plate containing 70% Sf9 insect cells; culture for 5 days and collect the supernatant to obtain the recombinant baculovirus vAcBacIIIG / rFC opt.

[0056] (4) Expression of FC opt

[0057] Add Sf9 insect cells to a 10 cm culture dish, infect the Sf9 insect cells with the collected recombinant baculovirus vAcBacIIIG / rFC opt, collect the supernatant and cell samples after 5 days of infection, and detect the expression level of FC opt by SDS-page.

[0058] (5) Detection of endotoxin sensitivity of FC opt

[0059] Perform protein sensitivity detection on the collected medium containing recombinant horseshoe crab factor C, that is, dilute the endotoxin standard product with endotoxin-free water to obtain endotoxin standard solutions of 0.5 EU / mL, 0.05 EU / mL and 0.005 EU / mL, add the fluorescent substrate solution, buffer solution and rFC opt enzyme solution (the medium expressing recombinant horseshoe crab factor C is used as the enzyme solution here) in a volume ratio of 5:4:1, mix well and add it to the enzyme-labeled plate with the endotoxin standard product added, with an excitation wavelength of 380 nm and an emission wavelength of 440 nm, read the fluorescence value; detect again after incubating at 37 °C for 1 h.

[0060] Comparative Example 1

[0061] In this comparative example, a certain company's codons were used to optimize the original gene sequence of horseshoe crab factor C, and the optimized mRNA sequence was scanned with the RNAfold program. It can be seen from the results that when the scanning window is 50 nucleotides in length, its free energy is similar to the original sequence of horseshoe crab factor C ( Figure 3 ). According to the optimized mRNA sequence, the optimized recombinant horseshoe crab factor C gene sequence (FC opt-H sequence) was obtained, as shown in SEQ ID NO:2.

[0062] The remaining steps of constructing the recombinant plasmid pQB3a-FC opt-H, constructing the recombinant baculovirus vAcBacIIIG / rFC opt-H, the expression of FC opt-H and the detection of endotoxin sensitivity of FC opt-H are the same as those in Example 1 and will not be repeated.

[0063] Figure 4To compare the protein expression of horseshoe crab Factor C (FC group), recombinant horseshoe crab Factor C after optimizing the method of Example 1 (FC opt group), and recombinant horseshoe crab Factor C after optimizing the method of Comparative Example 1 (FC opt-H group) by SDS-PAGE; it can be seen from the figure that the protein expression in the FC group is the least, the protein expression in the FC opt-H group is increased compared with that in the FC group, and the protein expression in the FC opt group is the highest.

[0064] Figure 5 To compare the endotoxin detection effects of horseshoe crab Factor C (FC group), recombinant horseshoe crab Factor C after optimizing the method of Example 1 (FC opt group), and recombinant horseshoe crab Factor C after optimizing the method of Comparative Example 1 (FC opt-H group); it can be seen from the sensitivity detection results that the endotoxin detection sensitivity of the FC opt-H group is similar to that of the FC group, and the endotoxin detection sensitivity of the FC opt group is higher.

[0065] In terms of codon optimization: The present invention sets different nucleotide length windows (30, 40, 50, 60 nt), scans the original mRNA sequence of Factor C with the RNAfold program, and obtains the minimum free energy (MEF) of RNA folding at different window lengths. It is found that when scanning with a 50-nucleotide length window, the minimum free energy can reach -20 kcal / mol( Figure 1 ). The present invention uses the mRNA sequence of horseshoe crab Factor C as the starting sequence and recodes horseshoe crab Factor C. Comparing with the original mRNA sequence, the optimized mRNA sequence: 1. Removes six rare codons (ctt, cta, cgt, cga, cgg, ggg); 2. For any 50-nucleotide length RNA fragment, the minimum free energy of folding is not less than -12 kcal / mol. Thus, the obtained mRNA has fewer secondary structures that are difficult to open, the movement hindrance of ribosomes is reduced, and the protein yield is increased.

[0066] In terms of recombinant proteins: Through the above codon optimization scheme, the present invention finally obtains a recombinant horseshoe crab Factor C gene sequence. Using the insect cell-baculovirus expression system to express the original gene sequence and the gene sequence after codon optimization, it can be seen by comparing the differences in protein expression levels that: the protein expression level after optimization by the above codon optimization scheme is much higher than that of the unoptimized protein and the proteins optimized by other codon optimization schemes; that is, the protein expression level of recombinant horseshoe crab Factor C is successfully increased through the codon optimization scheme of this project, and in terms of endotoxin detection, the recombinant horseshoe crab Factor C (FC opt) after codon optimization shows stronger sensitivity compared with the original horseshoe crab Factor C and the recombinant horseshoe crab Factor C (FC opt-H) obtained by other codon optimization schemes.

[0067] Finally, it should be noted that the above-described embodiments merely represent several implementation manners of the present invention and are not intended to limit the present invention. For those of ordinary skill in the art, any modifications, equivalent substitutions, improvements, etc. made without departing from the concept of the present invention shall be included within the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A codon-optimized recombinant horseshoe crab factor C, characterized in that: The gene sequence of the recombinant horseshoe crab factor C is shown in SEQ ID NO:

1.

2. A recombinant plasmid, characterized in that: The recombinant plasmid contains the recombinant horseshoe crab factor C according to claim 1.

3. A recombinant baculovirus, characterized in that The recombinant baculovirus contains the recombinant horseshoe crab factor C sequence according to claim 1.

4. A recombinant horseshoe crab factor C mRNA, characterized in that: The mRNA is transcribed from the gene sequence of the recombinant horseshoe crab factor C according to claim 1.

5. A recombinant horseshoe crab factor C protein, characterized in that: The recombinant horseshoe crab factor C protein is obtained by expressing the recombinant horseshoe crab factor C according to claim 1 in cells.

6. The recombinant horseshoe crab factor C protein according to claim 5, characterized in that The cells are insect cells Sf9 or HighFive.

7. A method for preparing the recombinant horseshoe crab factor C protein according to claim 5, characterized in that: The following steps are involved: Transfecting or infecting Sf9 or HighFive cells with the recombinant plasmid of claim 2 or the recombinant baculovirus of claim 3; Cultivating the Sf9 or HighFive cells; The culture medium was collected to obtain the recombinant horseshoe crab factor C protein.

8. An endotoxin detection reagent, characterized in that: Comprising the recombinant horseshoe crab factor C protein according to claim 5 or 6.

9. An endotoxin detection kit, characterized in that: Contains the endotoxin detection reagent according to claim 8.

10. A method for detecting endotoxin, characterized in that: Endotoxin detection is performed using the recombinant horseshoe crab factor C protein described in claim 5 or 6, the endotoxin detection reagent described in claim 8, or the endotoxin detection kit described in claim 9.

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