Polynucleotide fragments that enhance protein secretion and uses thereof

Abstract

This invention relates to the field of molecular biology, specifically to a polynucleotide fragment that enhances protein secretion and its applications. The polynucleotide fragment enhancing protein secretion comprises truncated fragments of the sequences shown in F162 and F516; or fragments having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the fragments shown in F162 and F516 and possessing protein secretion-enhancing function. This invention has screened a broad-spectrum, core-positively regulated polynucleotide fragment that can enhance protein secretion, significantly increasing protein secretion levels independently in multiple cell types including HEK293T, HeLa, and HUVEC. Simultaneously, the polynucleotide fragment of this invention exhibits a strong synergistic enhancement effect with signal peptides such as SPI and SPG, significantly improving the secretory expression level of the target protein, making it suitable for constructing efficient expression vectors.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a polynucleotide fragment that enhances protein secretion and its applications. Background Technology

[0002] Protein secretion is a crucial process in cell biology, regulated by multiple mechanisms. The most common is signal peptide-mediated extracellular secretion. Signal peptides are typically short peptides located at the N-terminus of proteins. For mature secretory proteins, a 15-30 amino acid signal peptide is synthesized at the N-terminus. This signal peptide guides the nascent polypeptide chain into the endoplasmic reticulum, where it is usually cleaved by a signal peptidase, ultimately releasing the mature protein extracellularly. Different types of secretory proteins exhibit significantly different secretion efficiencies under the influence of their respective signal peptides. For certain hematologic disorders and inflammatory diseases, therapeutic proteins need to be secreted extracellularly to exert their effects; therefore, increasing the expression and secretion levels of these proteins is essential.

[0003] In recent years, researchers have attempted to optimize protein secretion efficiency, for example, by screening for highly efficient signal peptides targeting specific proteins. One researcher screened eight signal peptides in Bacillus subtilis to optimize the secretion of mesophilic α-amylase (Zhang Shibin, Chen Jingqi, Cui Yanyan, et al. High-efficiency expression of mesophilic α-amylase gene in Bacillus subtilis and optimization of fermentation conditions [J]. Food and Fermentation Industries, 2016, 42(4): 50-56.). Studies have shown that different signal peptides have different effects on the same secretory protein, and the same signal peptide also has significantly different secretion effects on different proteins. There is a lack of universal compatibility rules between signal peptides and target proteins; the secretion effects of the same signal peptide on different proteins vary significantly; and there is no significant correlation between the sequence characteristics of the signal peptide and its secretion performance. This means that screening for the optimal signal peptide for a specific protein still requires large-scale experimental screening. Summary of the Invention

[0004] The purpose of this invention is to provide a polynucleotide fragment that enhances protein secretion and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A polynucleotide fragment that enhances protein secretion, comprising a truncated fragment of the sequence shown in F162 or F516; or a fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the fragments described in F162 or F516 and having the function of enhancing protein secretion.

[0007] The sequence of F162 is shown in SEQ ID NO.1;

[0008] The sequence of F516 is shown in SEQ ID NO.2.

[0009] Through extensive experimentation, the inventors of this invention unexpectedly discovered a polynucleotide fragment that can enhance protein secretion. This polynucleotide fragment, used alone or in combination with different signal peptides, significantly increases the secretion level of related proteins in different cells.

[0010] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0011] In one preferred embodiment, the polynucleotide fragment comprises a truncated fragment of the sequence shown in F516; or a fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the fragment shown in F516 and having the function of enhancing protein secretion.

[0012] Extensive experiments have demonstrated that F516 can independently exert a broad-spectrum enhancing effect in a variety of cell types.

[0013] Based on the same inventive concept, the present invention also claims a vector comprising any of the above-mentioned polynucleotide fragments.

[0014] In one preferred embodiment, the carrier comprises a promoter.

[0015] In one preferred embodiment, the promoter is located downstream of the polynucleotide fragment.

[0016] In one preferred embodiment, the polynucleotide fragment is adjacent to the promoter.

[0017] In one preferred embodiment, the promoter is selected from constitutive promoters, tissue-specific promoters, and inducible promoters.

[0018] In one preferred embodiment, the promoter is selected from the mini promoter, F8 promoter, CMV promoter, EF1α promoter, SV40 promoter, CAG promoter or PGK promoter.

[0019] In one preferred embodiment, the carrier comprises a signal peptide.

[0020] In one preferred embodiment, the signal peptide is located downstream of the promoter.

[0021] In one preferred embodiment, the signal peptide is selected from SPI, SPG, Qα, or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SPI, SPG, or Qα.

[0022] In one preferred embodiment, the signal peptide is selected from SPI, SPG, or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SPI or SPG; more preferably, the signal peptide is SPG or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SPG.

[0023] SPI and SPG have different effects in different cells, but F516 is a core broad-spectrum positive key element and has a conserved strong co-activation effect with SPI and SPG. This co-activation mode can play a role in both universal mini promoters and specific F8 promoters.

[0024] Furthermore, regardless of whether it is a universal promoter or an endothelial-specific F8 promoter, F516+SPG is the preferred combination for efficient expression.

[0025] In one preferred embodiment, the vector contains a foreign gene located downstream of the promoter.

[0026] In one preferred embodiment, the exogenous gene is one or more of luciferase, kinase, phosphatase, protease, oxidoreductase, or transferase; more preferably, the enzyme protein is Gaussia luciferase.

[0027] Based on the same inventive concept, the present invention also claims a kit comprising any of the above-described polynucleotide fragments or any of the above-described vectors.

[0028] Based on the same inventive concept, the present invention also claims protection for a recombinant cell comprising any of the above-described polynucleotide fragments or any of the above-described vectors.

[0029] Based on the same inventive concept, the present invention also claims protection for the use of the polynucleotide fragment, the vector, the kit, or the recombinant cells in enhancing protein secretion.

[0030] In one preferred embodiment, the protein is one or more of luciferase, kinase, phosphatase, protease, oxidoreductase, or transferase; more preferably, the enzyme protein is luciferase.

[0031] In one preferred embodiment, the host cells transfected by the polynucleotide fragment or the vector are HEK293T, HeLa, or HUVEC cells.

[0032] Based on the same inventive concept, the present invention also claims a method for enhancing protein secretion, comprising the steps of placing any of the aforementioned polynucleotide fragments upstream of a gene.

[0033] In one preferred embodiment, the polynucleotide fragment is located before the promoter of the gene.

[0034] In one preferred embodiment, the polynucleotide fragment is adjacent to the promoter.

[0035] In one preferred embodiment, a signal peptide is provided downstream of the promoter.

[0036] In one preferred embodiment, the signal peptide is selected from SPI, SPG, Qα, or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SPI, SPG, or Qα.

[0037] Compared with the prior art, the beneficial effects of the present invention are:

[0038] This invention screened and obtained a broad-spectrum, core-positively regulated polynucleotide fragment that can enhance protein secretion. It can significantly increase protein secretion levels independently in multiple cell types, including HEK293T, HeLa, and HUVEC. Furthermore, experimental data demonstrates that this polynucleotide fragment exhibits a strong synergistic enhancement effect with signal peptides such as SPI and SPG, with the combined expression activity increasing by up to 200-fold, far superior to using a single element alone. This significantly improves the secretory expression level of the target protein and is suitable for constructing high-efficiency expression vectors. Moreover, it can achieve stable synergistic activation across cell lines, providing a basis for customized element combinations for gene expression vectors in different cell types and scenarios. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the multinucleotide fragments, promoters, signal peptides, and downstream genes of the plasmid obtained in Example 1.

[0040] Figure 2 This is a bar graph showing the luciferase detection results of HEK293T cells.

[0041] Figure 3 This is a bar graph showing the luciferase detection results of HeLa cells.

[0042] Figure 4 This is a bar graph showing the luciferase detection results of HUVEC cells.

[0043] Figure 5 This is a bar graph showing the luciferase detection results of HUVEC cells transfected with the specific promoter F8. Detailed Implementation

[0044] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0045] Terminology Explanation:

[0046] In this document, the term "polynucleotide sequence" also includes the complementary sequence of the polynucleotide sequence, and the term "complementary sequence" as used herein has the meaning commonly understood by those skilled in the art.

[0047] In this paper, the vector is a eukaryotic cell expression vector that expresses the foreign gene it carries in the cell.

[0048] In this document, "downstream" has the meaning well known to those skilled in the art, referring to a polynucleotide fragment located at or before the 5' end of the promoter. As used herein, "promoter" refers to a DNA sequence capable of enabling gene transcription, which can be recognized by RNA polymerase and initiate transcription to synthesize RNA. All promoters known in the prior art can be used in this invention.

[0049] In this document, in some embodiments, there is no space between the promoter and the polynucleotide fragment; in other embodiments, there is a space between the promoter and the polynucleotide fragment, such as a space of 6-40 bp, 2-50 bp, 1-100 bp, or longer. The spacer sequence may be a multiple cloning site, a recombination site, or may contain a transcription factor binding site.

[0050] The term "constitutive promoter" in this article, also known as a universal promoter, refers to a promoter that can continuously initiate gene expression, usually derived from housekeeping genes or viral genes.

[0051] The term "tissue-specific promoter" in this article refers to a promoter that can initiate gene expression only in a specific organ (or tissue).

[0052] The term "inducible promoter" in this article refers to a promoter that can only initiate gene expression under certain external induction (or stimulation) conditions, such as the tetracycline promoter TRE.

[0053] The figures in this invention only indicate the significant differences in some data (ns = no significant difference, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). The significant differences in some data that are not significant or are lower than the control group are not marked.

[0054] Example 1

[0055] Polynucleotide fragments F162 and F516 were both synthesized by Shanghai Sangon Biotech Co., Ltd.

[0056] The nucleotide sequence of F162 is as follows:

[0057] AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCACTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGCTCTGCAAAGAAATTGGGA (SEQ ID NO. 1).

[0058] The nucleotide sequence of F516 is as follows:

[0059] CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATATTTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCATGGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGGGGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTGGGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTTTCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTG(SEQ IDNO.2)。

[0060] The pGL3-luciferase plasmid (GenBank® Accession Number U47298) was modified by replacing the SV40 promoter with a mini promoter and the F8 promoter through enzyme digestion and homologous recombination. The luciferase gene expression cassette in this plasmid was then replaced with a synthetic Gaussian luciferase gene expression cassette through enzyme digestion and homologous recombination. The constructed promoter-Gaussian luciferase plasmids were named mini-pGluc and F8-pGluc, respectively. Simultaneously, plasmids containing classic signal peptides were constructed. After synthesis, the signal peptide fragments were inserted after the promoter, resulting in the following sequences: Mini-Qα-pGluc, Mini-SPI-pGluc, Mini-SPG-pGluc, F8-Qα-pGluc, F8-SPI-pGluc, and F8-SPG-pGluc. Polynucleotide fragments obtained after PCR amplification were then inserted before the promoters of these plasmids already containing classic signal peptides, resulting in the following sequences: F516-Mini-Qα-pGluc, F516-Mini-SPI-pGluc, F516-Mini-SPG-pGluc, F516-F8-Qα-pGluc, F516-F8-SPI-pGluc, F516-F8-SPG-pGluc, F162-Mini-Qα-pGluc, F162-Mini-SPI-pGluc, and F162-Mini-SPG-pGluc. pGluc, F162-F8-Qα-pGluc, F162-F8-SPI-pGluc, F162-F8-SPG-pGluc.

[0061] The specific steps are as follows:

[0062] (1) Enzyme digestion of the backbone plasmid

[0063] 1) Prepare the enzyme digestion system on ice and incubate overnight at 37 °C for 12 h. The enzyme digestion system is as follows:

[0064]

[0065] 2) Purify the linearized vector and measure its concentration.

[0066] (2) Obtain the target segment

[0067] 1) Amplification was performed using Vazyme's 2 × phanta Max Master Mix. The reaction system is as follows:

[0068]

[0069] The reaction procedure is as follows:

[0070] .

[0071] 2) Recover the target ligation fragments from the gel, purify the product, and determine its concentration.

[0072] (3) Homologous recombination: Homologous recombination was performed using Vazyme's ClonExpress Ultra One Step Cloning Kit V2 (C116-01) according to the operating instructions:

[0073] 1) Design homologous recombination primers with homologous arms set at 20 bp;

[0074]

[0075] Genes without downstream primers also have downstream primers, all of which are GLUC-R.

[0076] 2) Calculate the amount of carrier and linker product used according to the following formula;

[0077] Single-segment homologous recombination reaction:

[0078] Optimal cloning vector usage = [0.02 × number of base pairs in cloning vector] ng (0.03 pmol).

[0079] Optimal amount of insert fragment used = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol).

[0080] To construct different promoter-Gaussian luciferase plasmids, namely mini-pGluc and F8-pGluc, simply replace the promoter with a mini promoter or an F8 promoter. In this case, it is a single-fragment homologous recombination, and the calculation is performed according to the formula for single-fragment homologous recombination reaction.

[0081] Multi-fragment homologous recombination reaction:

[0082] Optimal cloning vector usage = [0.02 × number of base pairs in cloning vector] ng (0.03 pmol).

[0083] Optimal amount of insert fragment used = [0.02 × number of base pairs per fragment] ng (0.03 pmol).

[0084] Construct plasmids containing classic signal peptides, namely Mini-Qα-pGluc, Mini-SPI-pGluc, Mini-SPG-pGluc, F8-Qα-pGluc, F8-SPI-pGluc, and F8-SPG-pGluc; and recombinant plasmids with polynucleotide fragments inserted before the promoters of these plasmids already containing classic signal peptides, namely F516-Mini-Qα-pGluc, F516-Mini-SPI-pGluc, F516-Mini-SPG-pGluc, F516-F8-Qα-pGluc, F516-F8-SPI-pGluc, F516-F8-SPG-pGluc, F162-Mini-Qα-pGluc, F162-Mini-SPI-pGluc, F162-Mini-SPG-pGluc, and F162-F8-Qα-pGluc. For pGluc, F162-F8-SPI-pGluc, and F162-F8-SPG-pGluc, the fragments to be inserted are signal peptide and promoter, polynucleotide sequence, and signal peptide and promoter. This is a multi-fragment homologous recombination, and the calculation is performed according to the formula for multi-fragment homologous recombination reaction.

[0085] 3) Prepare ice, prepare the system on the ice, and add sterile water to bring the volume to 10 µL;

[0086]

[0087] 4) For single-fragment ligation PCR, incubate at 50 ℃ for 5 minutes and immediately place on ice to cool after the reaction; for ligation PCR of 2-3 fragments, incubate at 50 ℃ for 15 minutes and immediately place on ice to cool after the reaction.

[0088] (4) Transformation

[0089] 1) Thaw DH5α competent cells on ice, add 5 µL of recombinant product to every 50 µL of competent cells, incubate on ice for 15 minutes, heat shock at 42 ℃ for 60 sec, and incubate on ice for 2 minutes.

[0090] 2) In a clean bench, add 900 µL of liquid LB, set the shaker parameters to 220 rpm, 37 ℃, shake for 30 minutes, centrifuge at 2000 rpm for 2 minutes, discard 800 µL of supernatant, and use 100 µL of resuspended bacterial solution for coating.

[0091] 3) Incubate in an inverted position in a 37 ℃ constant temperature incubator;

[0092] 4) Select single-clone colonies, set the shaker parameters to 220 rpm and 37 ℃, shake for 5 hours, dispense the bacterial solution in a clean bench and send it for Sanger sequencing. If the sequencing results match the theoretical sequence, extract plasmids for cell transfection.

[0093] In this embodiment, the distance between the inserted fragment and the promoter is 30-40 bp, which is the restriction enzyme / cloning site. The distance may vary slightly depending on the restriction enzyme site used, but it does not affect gene expression.

[0094] The sequence of the signal peptide is shown below.

[0095] The SPI sequence is as follows:

[0096] ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGTGGGC (SEQ ID NO. 19).

[0097] The SPG sequence is as follows:

[0098] ATGGGCGTGAAGGTGCTGTTTGCCCTGATTTGCATCGCCGTGGCCGAGGCC (SEQ ID NO. 20).

[0099] The sequence of Qα is as follows:

[0100] CAACCGCGGTTCGCGGCCGCT (SEQ ID NO. 21).

[0101] A schematic diagram illustrating the construction of plasmids containing multinucleotide fragments, promoters, signal peptides, and their downstream genes is shown below. Figure 1 As shown.

[0102] The plasmid sequence is shown below. The bold blue bases are the promoter sequence, the black bases are the linker sequence, the red underlined bases are the signal peptide sequence, and the gray bases are the Gluc expression cassette sequence.

[0103] The sequence of Mini-Qα-pGluc is as follows:

[0104] TAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTTATG CAACCGCGGTTCGCGGCCGCTGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.22).

[0105] The sequence of Mini - SPI - pGluc is as follows:

[0106] TAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTT ATGGCCTTGACCTTTGCTTTACTGGTGGCC CTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGTGGGCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.23).

[0107] The sequence of Mini-SPG-pGluc is as follows:

[0108] TAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTT ATGGGCGTGAAGGTGCTGTTTGCCCTGATT TGCATCGCCGTGGCCGAGGCCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.24).

[0109] The sequence of F516-mini-pGluc is as follows: CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATAT TTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACC TGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCAT GGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGG GGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTG GGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTT TCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTGGGTACCTTACGCGTGCTAGCCCGGGCTCGAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTTGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.25).

[0110] The sequence of F516-Mini-Qα-pGluc is as follows: CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATAT [[ID=*6]] TTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACC TGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCAT GGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGG GGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTG GGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTT TCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTG Note: There seems to be an asterisk added in the translation of line ID=3 in the provided text which might be an error. The original text doesn't have it. The above translation is based on the provided rules and the original text.GGTACCTTACGCGTGCTAGCCCGGGCTCGAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTTATG CAACCGCGGTTC GCGGCCGCT GGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.26).

[0111] The sequence of F516-Mini-SPI-pGluc is as follows: CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATAT TTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACC TGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCAT GGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGG GGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTG GGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTT TCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTG GGTACCTTACGCGTGCTAGCCCGGGCTCGAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTT ATGGCCTTGACCTTT GCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGTGGGC ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.27).

[0112] The sequence of F516-Mini-SPG-pGluc is as follows: CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATAT TTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACC TGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCAT GGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGG GGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTG GGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTT TCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTG GGTACCTTACGCGTGCTAGCCCGGGCTCGAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTT ATGGGCGTGAAGGTG CTGTTTGCCCTGATTTGCATCGCCGTGGCCGAGGCC ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.28).

[0113] The sequence of F162-mini-pGluc is as follows: AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCA CTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGC TCTGCAAAGAAATTGGGA GGTACCTTACGCGTGCTAGCCCGGGCTCGAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTTGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.29).

[0114] The sequence of F162-Mini-Qα-pGluc is as follows: AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCA CTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGC TCTGCAAAGAAATTGGGA GGTACCTTACGCGTGCTAGCCCGGGCTCGAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTTATG CAACCGCGGTTCGCGGCCGCT GGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.30).

[0115] The sequence of F162-Mini-SPI-pGluc is as follows: AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCA CTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGC TCTGCAAAGAAATTGGGA GGTACCTTACGCGTGCTAGCCCGGGCTCGAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTT ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGT GGGCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.31).

[0116] The sequence of F162-Mini-SPG-pGluc is as follows: AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCA CTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGC TCTGCAAAGAAATTGGGA GGTACCTTACGCGTGCTAGCCCGGGCTCGAGTAGAGGGTATATAATGGAAGCTCGACTTCCAGAAGCTT ATGGGCGTGAAGGTGCTGTTTGCCCTGATTTGCATCGCCGTGGCCGAGGCC ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.32).

[0117] The sequence of F8-Qα-pGluc is as follows: CAACCGCGGTTCGCGGCCGCT GGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.33).

[0118] The sequence of F8-SPI-pGluc is as follows: ATGGCCTTGACCTTTGCTTTACTGGTG GCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGTGGGC ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.34). <00​​ ATGGGCGTGAAGGTGCTGTTTGCCCTG ATTTGCATCGCCGTGGCCGAGGCC ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.35).

[0120] The sequence of F516-F8-pGluc is as follows: CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATAT TTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACC TGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCAT GGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGG GGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTG GGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTT TCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTG

[0121] The sequence of F516-F8-Qα-pGluc is as follows: CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATAT TTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACC TGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCAT GGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGG GGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTG GGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTT TCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTG CAACCGCGG TTCGCGGCCGCT GGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.37).

[0122] The sequence of F516-F8-SPI-pGluc is as follows: CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATAT TTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACC TGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCAT GGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGG GGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTG GGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTT TCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTG ATGGCCTTGACC TTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGTGGGC ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.38).

[0123] The sequence of F516 - F8 - SPG - pGluc is as follows: CAAAGAAATTGGGACTTTTCATTAAATCAGAAATTTTACTTTTTTCCCCTCCTGGGAGCTAAAGATAT TTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAGCAATAAGTCATGCAAATAGAGCTCTCCACC TGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCAT GGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGGTAAAGGCATGTCCTGTAGGGTCTGATCGG GGCCAGGATTGTGGGGATGTAAGTCTGCTTGGAGGAAGGTGCAGACATCGGGTTAGGATGGTTGTGATGCTACCTG GGCCCCAAAGAAACATTTCTGGGTAAGGTGTGCACACATCTGTGTTATTAGCAGAAATGCTAACTGCCAATTCTTT TCATAGGTCTGACCTATTTGTTGATATTTTGTTCTGTTTTGTCCATTGCTTCTCTTCGTCATATGCTG ATGGGCGTGAAG GTGCTGTTTGCCCTGATTTGCATCGCCGTGGCCGAGGCC ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.39).

[0124] The sequence of F162 - F8 - pGluc is as follows: AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCA CTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGC TCTGCAAAGAAATTGGGA

[0125] The sequence of F162-F8-Qα-pGluc is as follows: AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCA CTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGC TCTGCAAAGAAATTGGGA CAACCGCGGTTCGCGGCCGCT GGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.41).

[0126] The sequence of F162 - F8 - SPI - pGluc is as follows: AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCA CTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGC TCTGCAAAGAAATTGGGAATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTC TGTGGGC ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGCGGCCGCAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTC (SEQ ID NO.42).

[0127] The sequence of F162-F8-SPG-pGluc is as follows: AGAAGTGAATGGGTTAAGTTTAGCAGCCTCCCTTTTGCTACTTCAGTTCTTCCTGTGGCTGCTTCCCA CTGATAAAAAGGAAGCAATCCTATCGGTTACTGCTTAGTGCTGAGCACATCCAGTGGGTAAAGTTCCTTAAAATGC TCTGCAAAGAAATTGGGAATGGGCGTGAAGGTGCTGTTTGCCCTGATTTGCATCGCCGTGGCCGAGGCC (SEQ ID NO.43).

[0128] Example 2

[0129] The constructed Luciferase reporter vector was transfected into the cell line.

[0130] Experiments were conducted in three cell lines: HeLa (human cervical cancer cell line), HUVEC (human umbilical vein endothelial cell line), and HEK-293T (human embryonic kidney cell line) to verify the effects of different inserted polynucleotide fragments, alone or in combination with different signal peptides, on the secretion levels of reporter gene expression proteins. Previous experiments showed that the F8 promoter only functions effectively in HUVECs; therefore, all F8 promoter-related plasmids were used only in HUVEC cells.

[0131] Cells were digested into single cells using trypsin digestion and counted using a hemocytometer. 100,000 cells were seeded into each well (using a 48-well plate as an example). After cross-linking, the cell culture plate was placed in a humidified incubator at 37°C and 5% CO2. Transfection was performed when the confluence of the seeded cells reached approximately 80%. The medium was changed 2 hours before transfection. Transfection was performed using HieffTrans® Liposomal 2000 Transfection Reagent (Yisheng, 40802ES01) according to the instruction manual. The internal control plasmid pCMV-Cypridina Luc (Novopro, Accession Number V011557) and the Gaussian fluorescent reporter plasmid were co-transfected. Taking the Mini promoter group as an example, 12 groups were set up: mini-pGluc, F516-mini-pGluc, F162-mini-pGluc, Mini-Qα-pGluc, Mini-SPI-pGluc, Mini-SPG-pGluc, F516-Mini-Qα-pGluc, F516-Mini-SPI-pGluc, F516-Mini-SPG-pGluc, F162-Mini-Qα-pGluc, F162-Mini-SPI-pGluc, and F162-Mini-SPG-pGluc. The plasmid transfection amount for each group was 150 ng / well, and all were co-transfected with 10 ng / well of pCMV-Cypridina Luc internal control plasmid. After 12 hours of transfection, the old culture medium was discarded and replaced with fresh culture medium for further culture. After 48 hours of transfection, the cell supernatant was collected for Gaussian culture. Luciferase (Gaussia luciferase) reporter assay. Each experiment was independently repeated three times.

[0132] For HeLa and HEK-293T cell lines, Hieff Trans® Liposomal 2000 Transfection Reagent (Yisheng, 40802ES01) was used for transfection, while HUVEC was transfected using FuGENE® 4K Transfection Reagent (Promega, E5911).

[0133] Luciferase assay

[0134] Cell culture supernatant was collected 48 hours after transfection and tested using Cypridina Luciferin from Nanolight Technology, following the instructions. Luciferase activity was detected using a SpectraMax iD3 multi-functional microplate reader to screen for broad-spectrum and highly efficient components and combinations of components that enhance protein secretion.

[0135] The luciferase assay results of HEK293T cells are as follows: Figure 2 As shown in the figure. The results showed that in HEK293T cells, the activities of most groups among mini-pGluc, Mini-Qa-pGluc, Mini-SPI-pGluc, Mini-SPG-pGluc, F162-mini-pGluc, F162-Mini-Qa-pGluc, and F516-Mini-Qa-pGluc were extremely low, and the differences between groups were ns (not statistically significant) or small, indicating that these unit components / basic combinations have no transcriptional driving activity in HEK293T cells and have extremely low background activity.

[0136] The activities of F162-Mini-SPI-pGluc and F162-Mini-SPG-pGluc were slightly higher than those of the control group.

[0137] The F516-mini-pGluc activity was significantly higher than that of the control group, making it a variant with an enhancing effect among the unit components. This indicates that the polynucleotide fragment F516 alone can significantly increase luciferase activity in cell supernatants, with a higher enhancement effect than using the classic signal peptides SPI or SPG alone.

[0138] The combination of F516 with signal peptides SPI or SPG, resulting in F516-Mini-SPI-pGluc and F516-Mini-SPG-pGluc, further increased protein secretion levels by up to 130-200 times, significantly higher than the control group and other variants. Both combinations also far exceeded the activity levels of all other combinations. This suggests a strong synergistic effect between F516 and SPI / SPG, enabling efficient enhancement of protein secretion in HEK293T cells.

[0139] The results of luciferase assay in HeLa cells are as follows: Figure 3As shown in the figure. The results showed that the activities of mini-pGluc, Mini-Qα-pGluc, Mini-SPI-pGluc, and F162-Mini-Qα-pGluc were all very low in HeLa cells, with differences between groups of only ns. This indicates that the truncated mini promoters also have extremely low background activity in HeLa cells, and that the Qα and SPI elements have no additional regulatory effect. The activity of Mini-SPG-pGluc was significantly higher than that of the control group, and it was the sequence with the strongest enhancing effect among the unit components, indicating that the SPG element has strong enhancing activity in HeLa cells. The activities of F162-mini-pGluc and F516-mini-pGluc were significantly higher than those of the control group, but lower than those of Mini-SPG-pGluc. The enhancing effect was weakened when F162 was combined with Qα, indicating a certain antagonistic effect. The activities of F162-Mini-SPI-pGluc and F162-Mini-SPG-pGluc were significantly higher than those of the control group and slightly higher than those of F162-mini-pGluc, indicating a certain synergistic effect. The activity of F516-Mini-Qα-pGluc was not significantly different from that of F516-mini-pGluc, indicating that Qα could not regulate the enhancing function of F516. However, the activities of F516-Mini-SPI-pGluc and F516-Mini-SPG-pGluc were the highest among all groups, significantly higher than those of the control group and other variants, exhibiting a very strong synergistic enhancing effect and representing the optimal combination for efficient secretion of driver genes in HeLa cells.

[0140] Following a similar procedure, a series of plasmids containing different polynucleotide fragments and different promoters were transfected into HUVEC cell lines. The results are as follows: Figure 4As shown, the activities of mini-pGluc and Mini-Qα-pGluc were low, with inter-group differences of only ns (not statistically significant), indicating that the truncated mini promoters have extremely low background activity in HUVECs, and the Qα element has no additional enhancing effect. The activities of Mini-SPI-pGluc and Mini-SPG-pGluc were significantly higher than those of the control group, indicating that the SPI element can significantly enhance the luciferase secretion activity driven by the mini promoter, and the enhancement effect of SPG is better than that of SPI. The activity of F162-mini-pGluc was not significantly different from that of the control group and did not have independent enhancement ability. Compared with reporter gene expression driven solely by mini promoters, F516-mini-pGluc can significantly increase protein secretion levels in HUVECs and stably exert an enhancing effect. The activities of F162-Mini-Qα-pGluc, F162-Mini-SPI-pGluc, and F162-Mini-SPG-pGluc were significantly higher than those of the control group. However, F162-Mini-Qα-pGluc and F162-Mini-SPI-pGluc showed no significant improvement compared to F162-mini-pGluc, indicating that Qα and SPI did not further enhance activity. The enhancement effect of F162-Mini-SPG-pGluc was better than that of the F162 unit, indicating a certain synergistic effect between F162 and SPG. The activity of F516-Mini-Qa-pGluc was significantly lower than that of F516-mini-pGluc, indicating that the addition of the Qα sequence weakened the enhancement effect of F516-mini. The activity of F516-Mini-SPI-pGluc was the highest among all groups, significantly higher than that of the control group and other variants, indicating that the combination of F516 and SPI has a very strong synergistic enhancement effect, driving an activity increase far greater than that of the unit. The activity of F516-Mini-SPG-pGluc was also high, significantly higher than that of the control group and most variants. Its enhancement effect was second only to F516-Mini-SPI-pGluc, indicating that F516 and SPG also have a significant synergistic effect, but it is weaker than the combination with SPI.

[0141] Building upon the previous work, this invention aims to evaluate whether these enhancing elements, in addition to binding to universal promoters to exert enhancing effects, can also exert enhancing effects against the specific promoter F8. The constructed F8-pGluc series plasmids, containing insertions of different polynucleotide fragments, were transfected into HUVEC cells. The results are as follows... Figure 5As shown, compared to the control group F8-pGluc, the activities of F8-Qα-pGlu, F8-SPI-pGluc, F8-SPG-pGluc, F162-F8-pGluc, F162-F8-Qα-pGluc, F162-F8-SPI-pGluc, F162-F8-SPG-pGluc, and F516-F8-Qα-pGluc showed no significant difference or increase, while Qα exhibited a significant inhibitory effect. The activities of both F8-Qα-pGluc alone and the combination of F516-F8-Qα-pGluc were significantly lower than their corresponding basal groups. The addition of the Qα sequence weakened the enhancing effect of F516-F8. Compared to reporter gene expression and secretion driven solely by F8, the activity of F516-F8-pGluc was significantly higher than the control group (approximately 1.25), a statistically significant difference, indicating that F516 can independently increase protein secretion levels. This indicates that F516 significantly enhances F8-driven protein secretion. While the SPI and SPG sequences had no significant effect on the activity of the basal F8 promoter, plasmids F516-F8-SPI-pGluc and F516-F8-SPG-pGluc, which combine the F516 signal peptide SPI or SPG, also significantly enhanced F8-driven protein secretion. This demonstrates that the F516 sequence is a key positive regulatory element, and its activity is further enhanced when combined with the SPG sequence (F516-F8-SPG-pGluc), making it the most potent driver. This suggests a synergistic enhancement effect between F516 and SPG, which can significantly improve the secretion capacity of F8 promoter-driven proteins in HUVEC cells.

[0142] The results of the above examples show that F162 has weak enhancing ability, limited applicability, and is prone to antagonism with other components. However, the F516 fragment has a significant effect on enhancing the expression and secretion of target genes, independently exerting a broad-spectrum enhancing effect in various cell types. It can enhance the expression and secretion of target genes by binding to both universal and specific promoters. The F516 fragment binds to the signal peptides SPI or SPG, exhibiting a conserved and strong co-activation effect. This co-activation mode can function in both universal mini promoters and specific F8 promoters, further enhancing protein secretion levels.

[0143] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A polynucleotide fragment that enhances protein secretion, characterized in that, It contains truncated fragments of the sequences shown in F162 and F516; or fragments that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the fragments described in F162 and F516 and have the function of enhancing protein secretion. The sequence of F162 is shown in SEQ ID NO.1; The sequence of F516 is shown in SEQ ID NO.

2.

2. A vector, characterized in that, It contains the polynucleotide fragment as described in claim 1.

3. The carrier according to claim 2, characterized in that, The vector contains a promoter; preferably, the promoter is located downstream of the polynucleotide fragment.

4. The carrier of claim 2, wherein, The promoter is selected from constitutive promoters, tissue-specific promoters, and inducible promoters; preferably, the promoter is selected from mini promoters, F8 promoters, CMV promoters, EF1α promoters, SV40 promoters, CAG promoters, or PGK promoters.

5. The carrier of claim 2, wherein, The vector contains a signal peptide; preferably, the signal peptide is located downstream of the promoter.

6. The carrier of claim 5, wherein, The signal peptide is selected from SPI, SPG, Qα or a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SPI, SPG or Qα.

7. The carrier of claim 2, wherein, The vector contains a foreign gene located downstream of the promoter; preferably, the foreign gene is one or more of luciferase, kinase, phosphatase, protease, oxidoreductase, or transferase; more preferably, the enzyme protein is Gaussia luciferase.

8. A reagent kit, characterized in that, It comprises the polynucleotide fragment of claim 1 or the vector of any one of claims 2-7.

9. A recombinant cell, characterized in that, It comprises the polynucleotide fragment of claim 1 or the vector of any one of claims 2-7.

10. The use of the polynucleotide fragment of claim 1, the vector of any one of claims 2-7, the kit of claim 8, or the recombinant cells of claim 9 in enhancing protein secretion.

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