Human transferrin fusion proteins and methods of making the same

Abstract

The present application discloses a human transferrin fusion protein and a preparation method thereof. In the present application, the fusion protein comprises: a human transferrin polypeptide element; and a signal peptide polypeptide element connected to one end of the human transferrin polypeptide element; wherein the amino acid sequence of the signal peptide polypeptide element is shown as SEQ ID NO: 4. The human transferrin fusion protein provided by the present application has the characteristics of high expression amount and high activity on a CHO vector, and has even higher antigenicity than natural human transferrin, and is suitable for scale preparation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to human transferrin fusion protein and its preparation method. Background Technology

[0002] Human transferrin (TRF) is a monomeric glycoprotein that binds iron. Its main physiological functions are binding and transporting ferric ions, controlling the concentration of free iron ions in body fluids, providing available iron, and preventing iron deposition in the blood. Mature transferrin consists of 679 amino acids with a molecular weight of approximately 80 kDa and can be divided into two domains with similar properties: TFN and TFC.

[0003] Plasma transferrin (TRF) levels can be used for the diagnosis of anemia and for monitoring treatment. In iron-deficiency hypochromic anemia, TRF levels are elevated (due to increased synthesis), but iron saturation is low (normal range 30%-38%). Conversely, if the anemia is due to impaired iron utilization by red blood cells (e.g., aplastic anemia), plasma TRF levels are normal or low. TRF often decreases during acute phase reactions, and therefore frequently decreases along with albumin and prealbumin in cases of inflammation and malignancy. Furthermore, it decreases in chronic liver disease and malnutrition, and can also serve as an indicator of nutritional status. However, monitoring human transferrin to obtain information about human health requires...

[0004] In existing technologies, the main methods for preparing human transferrin are natural extraction and in vitro amplification using constructed expression vectors. Natural extraction is difficult due to the scarcity of sources, cumbersome extraction steps, and unstable quality and activity. While the existing method of preparing human transferrin by constructing expression vectors for in vitro amplification solves the source problem and simplifies the preparation steps, it still suffers from low yield, poor stability, and low protein activity, making it difficult to prepare human transferrin on a large scale.

[0005] Therefore, there is still a need in this field to develop an efficient and stable method for preparing human transferrin. Summary of the Invention

[0006] The purpose of this invention is to provide a fusion protein.

[0007] Another object of the present invention is to provide a polynucleotide encoding the above-mentioned fusion protein.

[0008] Another object of the present invention is to provide an expression vector containing the above-mentioned polynucleotides.

[0009] Another object of the present invention is to provide a host cell containing the above-described expression vector.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned fusion protein.

[0011] Another object of the present invention is to provide a kit containing the above-mentioned fusion protein.

[0012] To address the aforementioned technical problems, the first aspect of the present invention provides a fusion protein, the fusion protein comprising:

[0013] Human transferrin polypeptide element; and

[0014] A signal peptide polypeptide element attached to one end of the human transferrin polypeptide element;

[0015] The amino acid sequence of the signal peptide polypeptide element is shown in SEQ ID NO:4.

[0016] In some preferred embodiments, the signal peptide element is attached to the N-terminus of the human transferrin.

[0017] The connection should be understood as a key connection or a sequence connection; in some preferred embodiments, the connection is a key connection.

[0018] In some preferred embodiments, the human transferrin polypeptide element is selected from any of the following:

[0019] (i) A polypeptide having the amino acid sequence shown in SEQ ID NO:1;

[0020] (ii) a polypeptide having ≥90% homology to the amino acid sequence shown in SEQ ID NO:1; and said polypeptide retaining the activity of the polypeptide shown in SEQ ID NO:1; and

[0021] (iii) A derivative polypeptide formed by substituting, deleting or adding 1 to 5 amino acid residues of the amino acid sequence shown in SEQ ID NO:1, and retaining the activity of the polypeptide with the amino acid sequence shown in SEQ ID NO:1.

[0022] In some preferred embodiments, the polypeptide having ≥90% homology with the amino acid sequence shown in SEQ ID NO:1 is preferably a polypeptide having ≥95% homology with the amino acid sequence shown in SEQ ID NO:1, more preferably a polypeptide having ≥97% homology with the amino acid sequence shown in SEQ ID NO:1, and even more preferably a polypeptide having ≥99% homology with the amino acid sequence shown in SEQ ID NO:1.

[0023] In some preferred embodiments, the other end of the human transferrin polypeptide element is attached to a (His)6 tag.

[0024] In some preferred embodiments, the human transferrin polypeptide element has a (His)6 tag attached to its C-terminus.

[0025] To avoid tag removal and secondary purification, and to obtain high-purity, high-activity target protein in one step, in some preferred embodiments, only the C-terminus of the human transferrin polypeptide element is linked with a (His)6 tag.

[0026] The amino acid sequence of SEQ ID NO:1 above is as follows:

[0027] VPDKTVRWCAV SEHEATKCQS FRDHMKSVIP SDGPSVACVK KASYLDCIRA IAANEADAVTLDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVV KKDSGFQMNQ LRGKKSCHTG LGRSAGWNIPIGLLYCDLPE PRKPLEKAVA NFFSGSCAPC ADGTDFPQLC QLCPGCGCST LNQYFGYSGA FKCLKNGAGDVAFVKHSTIF ENLANKADRD QYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKED LIWELLNQAQEHFGKDKSKE FQLFSSPHGK DLLFKDSAHG FFKVPPRMDA KMYLGYEYVT AIRNLREGTC QEAPTDECKPVKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKI MNGEADAMSL DGGFVYIAGK CGLVPVLAENYNKSDNCEDT PEAGYFAVAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWN IPMGLLYNKI NHCRFDEFFSEGCAPGSKKD SSLCKLCMGS GLNLCEPNNK EEGGGYTGAF RCLVEKGDVA FVKHQTVPQN TGGKNPDPWAKNLNEKDYEL LCLDGTRKPV EEYANCHLAR APNHAVVTRK DKEACVHKIL RQQQHLFGSN VTDCSGNFCLFRSETKDLLF RDDTVCLAKL HDRNTYEKYL GEEYVKAVGN LRKCSTSSLL EACTFRRP

[0028] The amino acid sequence of SEQ ID NO:4 above is as follows:

[0029] SEQ ID NO:4:

[0030] MKWVTFISLLFSSAYS

[0031] A second aspect of the present invention provides a codon-optimized polynucleotide encoding the fusion protein described in the first aspect of the present invention.

[0032] In some preferred embodiments, the polynucleotide is selected from any of the following:

[0033] (a) A polynucleotide having the nucleotide sequence shown in SEQ ID NO.2;

[0034] (b) A polynucleotide having ≥95% homology to the nucleotide sequence shown in SEQ ID NO.2; and

[0035] (c) Having a polynucleotide complementary to the polynucleotide sequence described in (a) or (b).

[0036] In some preferred embodiments, the polynucleotide having ≥95% homology with the nucleotide sequence shown in SEQ ID NO.2 is a polynucleotide having ≥97% homology with the nucleotide sequence shown in SEQ ID NO.2, more preferably, is a polynucleotide having ≥99% homology with the nucleotide sequence shown in SEQ ID NO.2.

[0037] In some preferred embodiments, the polynucleotide is selected from any of the following:

[0038] (a) A polynucleotide having the nucleotide sequence shown in SEQ ID NO. 6;

[0039] (b) A polynucleotide having ≥95% homology to the nucleotide sequence shown in SEQ ID NO. 6; and

[0040] (c) Having a polynucleotide complementary to the polynucleotide sequence described in (a) or (b).

[0041] The nucleotide sequence of SEQ ID NO.2 above is as follows:

[0042]

[0043] A third aspect of the present invention provides an expression vector containing the polynucleotide described in the second aspect of the present invention.

[0044] A fourth aspect of the present invention provides a host cell containing the expression vector described in the third aspect of the present invention.

[0045] In some preferred embodiments, the host cell is a eukaryotic cell; more preferably, the host cell is a mammalian cell; even more preferably, the host cell is a CHO cell.

[0046] The fifth aspect of the present invention provides a method for preparing a fusion protein, the method comprising the steps of:

[0047] Under suitable expression conditions, the host cells described in the fourth aspect of this invention are cultured to express the target protein; and

[0048] The target protein was isolated.

[0049] The suitable expression conditions described in this invention are those known in the art for culturing host cells.

[0050] The sixth aspect of the present invention provides a kit containing the fusion protein of the first aspect of the present invention, the polynucleotide of the second aspect of the present invention, the expression vector of the third aspect of the present invention, or the host cell of the fourth aspect of the present invention.

[0051] Compared with the prior art, the present invention has at least the following advantages:

[0052] (1) This invention constructs a preparation system for high-efficiency expression of human transferrin, which simulates the expression relief of natural human transferrin to obtain recombinant human transferrin with structure and biological function close to that of natural human transferrin;

[0053] (2) This invention obtains a transient expression vector with high expression levels by optimizing synonymous codon preference and screening signal peptides, thereby improving the expression efficiency of human transferrin;

[0054] (3) The method provided by the present invention can stably express human transferrin, and the prepared human transferrin fusion protein has good activity and its binding effect with antibodies is even better than that of natural protein.

[0055] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0056] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0057] Figure 1 This is a graph obtained by detecting the amount of target protein secreted in the supernatant of CHO cells using an enzyme-linked immunosorbent assay (ELISA) reader according to an embodiment of the present invention;

[0058] Figure 2 This is an electrophoresis diagram of the product expressed according to an embodiment of the present invention;

[0059] Figure 3 This is a diagram obtained by detecting antigenicity using an enzyme-linked immunosorbent assay (ELISA) reader according to an embodiment of the present invention. Detailed Implementation

[0060] Through extensive experimental research, the inventors have developed a human transferrin fusion protein that exhibits high expression levels and high activity on the CHO vector. Its antigenicity is even higher than that of natural human transferrin, making it suitable for large-scale preparation.

[0061] In one embodiment of the present invention, a fusion protein is provided, the fusion protein comprising:

[0062] Human transferrin polypeptide element; and

[0063] A signal peptide polypeptide element attached to one end of the human transferrin polypeptide element;

[0064] The amino acid sequence of the signal peptide polypeptide element is shown in SEQ ID NO:4.

[0065] In some preferred embodiments, the signal peptide element is attached to the N-terminus of the human transferrin.

[0066] The connection should be understood as a key connection or a sequence connection; in some preferred embodiments, the connection is a key connection.

[0067] In some preferred embodiments, the human transferrin polypeptide element is selected from any of the following:

[0068] (i) A polypeptide having the amino acid sequence shown in SEQ ID NO:1;

[0069] (ii) a polypeptide having ≥90% homology to the amino acid sequence shown in SEQ ID NO:1; and said polypeptide retaining the activity of the polypeptide shown in SEQ ID NO:1; and

[0070] (iii) A derivative polypeptide formed by substituting, deleting or adding 1 to 5 amino acid residues of the amino acid sequence shown in SEQ ID NO:1, and retaining the activity of the polypeptide with the amino acid sequence shown in SEQ ID NO:1.

[0071] In some preferred embodiments, the polypeptide having ≥90% homology with the amino acid sequence shown in SEQ ID NO:1 is preferably a polypeptide having ≥95% homology with the amino acid sequence shown in SEQ ID NO:1, more preferably a polypeptide having ≥97% homology with the amino acid sequence shown in SEQ ID NO:1, and even more preferably a polypeptide having ≥99% homology with the amino acid sequence shown in SEQ ID NO:1.

[0072] In some preferred embodiments, the other end of the human transferrin polypeptide element is attached to a (His)6 tag.

[0073] In some preferred embodiments, the human transferrin polypeptide element has a (His)6 tag attached to its C-terminus.

[0074] To avoid tag removal and secondary purification, and to obtain high-purity, high-activity target protein in one step, in some preferred embodiments, only the C-terminus of the human transferrin polypeptide element is linked with a (His)6 tag.

[0075] The amino acid sequence of SEQ ID NO:1 above is as follows:

[0076] VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQ LCQLCPGCGCSTLNQYFGYSGAFKCLKNGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFFKVPPRMDAKMYLGYEYVTAIRNLREGTCQEAPTDEC KPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAVAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPN NKEEGGGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP

[0077] SEQ ID NO:4:

[0078] MKWVTFISLLFSSAYS

[0079] Another embodiment of the present invention provides a codon-optimized polynucleotide encoding the fusion protein described in the first aspect of the present invention.

[0080] In some preferred embodiments, the polynucleotide comprises a polynucleotide sequence selected from any of the following:

[0081] (a) A polynucleotide having the nucleotide sequence shown in SEQ ID NO.2;

[0082] (b) A polynucleotide having ≥95% homology to the nucleotide sequence shown in SEQ ID NO.2; and

[0083] (c) Having a polynucleotide complementary to the polynucleotide sequence described in (a) or (b).

[0084] In some preferred embodiments, the polynucleotide having ≥95% homology with the nucleotide sequence shown in SEQ ID NO.2 is a polynucleotide having ≥97% homology with the nucleotide sequence shown in SEQ ID NO.2, more preferably, is a polynucleotide having ≥99% homology with the nucleotide sequence shown in SEQ ID NO.2.

[0085] The nucleotide sequence of SEQ ID NO.2 above is as follows:

[0086]

[0087] In some preferred embodiments, the polynucleotide is selected from any of the following:

[0088] (a) A polynucleotide having the nucleotide sequence shown in SEQ ID NO. 6;

[0089] (b) A polynucleotide having ≥95% homology to the nucleotide sequence shown in SEQ ID NO. 6; and

[0090] (c) Having a polynucleotide complementary to the polynucleotide sequence described in (a) or (b).

[0091] In another embodiment of the present invention, an expression vector is provided, the expression vector containing the polynucleotide described in the second aspect of the present invention.

[0092] In another embodiment of the present invention, a host cell is provided, the host cell containing the expression vector described in the third embodiment of the present invention.

[0093] In some preferred embodiments, the host cell is a eukaryotic cell; more preferably, the host cell is a mammalian cell; even more preferably, the host cell is a CHO cell.

[0094] Another embodiment of the present invention provides a method for preparing a fusion protein, the method comprising the steps of:

[0095] Under suitable expression conditions, the host cells described in the fourth aspect of this invention are cultured to express the target protein; and

[0096] The target protein was isolated.

[0097] The suitable expression conditions described in this invention are those known in the art for culturing host cells.

[0098] Another embodiment of the present invention provides a kit containing the fusion protein described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention.

[0099] As used herein, the term "fusion protein" is used interchangeably with "recombinant protein" and refers to a molecule in which two or more proteins or fragments thereof are covalently linked and contained in the backbone of each peptide. Fusion proteins are preferably produced through the genetic expression of polynucleotide molecules encoding these proteins.

[0100] As used in this article, the term “peptide” refers to a molecule containing a sequence of amino acids linked by peptide bonds, regardless of length, post-translational modifications, or function.

[0101] As used herein, the terms “peptide” and “peptide element” are used interchangeably to refer to proteins that are naturally occurring or that are produced or altered chemically or otherwise through recombination.

[0102] As used in this article, the term "signal peptide" refers to an RNA region following the start codon that encodes a sequence of hydrophobic amino acids. This amino acid sequence is called the signal peptide sequence and is responsible for guiding proteins into subcellular organelles containing different membrane structures.

[0103] As used herein, the terms "polynucleotide" and "polynucleotide sequence" can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding.

[0104] This invention also relates to variants of the aforementioned polynucleotides that encode protein fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded polypeptide.

[0105] As used herein, the terms “homology” and “sequence identity” are used interchangeably and refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by arranging the nucleotide or amino acid sequences of the polynucleotide or polypeptide, scoring the number of positions in the arranged polynucleotide or polypeptide containing the same nucleotide or amino acid residue, and comparing this to the number of positions in the arranged polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides can differ at one position, for example, by containing different nucleotides (i.e., substitutions or variations) or by the deletion of nucleotides (i.e., the insertion or deletion of one or two nucleotides in the polynucleotide). Polypeptides can differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or by the deletion of amino acids (i.e., the insertion of one or two amino acids in the polypeptide or the deletion of amino acids). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0106] As used herein, the terms “complementary sequence” or “reverse complementary sequence” are used interchangeably and refer to a sequence that is in the opposite direction to the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complementary sequence is GTTCAT.

[0107] As used in this article, the term "codon optimization" refers to the method of improving gene synthesis efficiency by avoiding the use of low-utilization or rare codons based on the differences in codon utilization exhibited by the actual organisms performing protein expression or production (including E. coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

[0108] As used herein, the term "vector" refers to a polynucleotide delivery vector. In some embodiments, in genetic engineering recombination techniques, a vector comprises a polynucleotide sequence encoding a specific protein that can be operatively inserted to achieve the expression of that protein. Vectors are used to transform, transduce, or transfect host cells and can express genetic material elements delivered by the vector in host cells. The term "vector" as used in this disclosure can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (comprising nucleic acid sequences comprising a series of suitable expression control elements). For example, a vector can be a recombinant plasmid vector, a recombinant eukaryotic viral vector, a recombinant bacterial bacteriophage vector, a recombinant yeast mini-chromosome vector, a recombinant bacterial artificial chromosome vector, or a recombinant yeast plasmid vector.

[0109] As used herein, the term "host cell" is a cell in which exogenous polynucleotides and / or vectors have been introduced. The host cell is a eukaryotic host cell or a prokaryotic host cell. The eukaryotic host cell can be a mammalian host cell, insect host cell, plant host cell, fungal host cell, eukaryotic algal host cell, nematode host cell, protozoan host cell, or fish host cell. For example, the host cell disclosed in this invention is a eukaryotic host cell, and the eukaryotic host cell is a mammalian host cell. The mammalian host cell is composed of Chinese hamster ovary cells (CHO cells), COS cells, Vero cells, SP2 / 0 cells, NS / O myeloid cells, human fetal kidney cells, immature hamster kidney cells, HeLa cells, human B cells, cv-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, or PerC6 cells. For example, the mammalian host cell in this disclosure is a CHO cell.

[0110] Preparation of fusion proteins

[0111] In this invention, the full-length nucleotide sequence or fragments of the fusion protein or its elements can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0112] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0113] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0114] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0115] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors or fusion protein coding sequences of the present invention, and methods for generating the proteins of the present invention via recombinant technology.

[0116] Using conventional recombinant DNA techniques, the polynucleotide sequence of this invention can be used to express or produce recombinant proteins. Generally, the following steps are involved:

[0117] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0118] (2) Host cells cultured in a suitable culture medium;

[0119] (3) Isolate and purify proteins from culture media or cells.

[0120] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0121] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0122] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0123] The host cell is preferably a eukaryotic cell, more preferably a higher eukaryotic cell, such as a mammalian cell. Representative examples include animal cells such as CHO, NSO, COS7, or 293 cells.

[0124] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0125] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0126] The proteins described in the above methods may be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0127] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0128] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0129] Example 1: Synonymous Codon Preference Optimization

[0130] Using the human TRF gene provided by NCBI as a reference, the sequence shown in SEQ ID NO:1 was optimized for synonymous codon bias after amino acid sequence analysis to determine the nucleotide sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, which were then synthesized by Nanjing GenScript. SEQ ID NO:5 contains the coding sequence for the signal peptide shown in SEQ ID NO:3, SEQ ID NO:6 contains the coding sequence for the albumin signal peptide (SEQ ID NO:4), and SEQ ID NO:7 contains the coding sequence for the α-factor signal peptide.

[0131] The clones were then inserted into the vector pcDNA3.1, with a (His)6 tag added to the C-terminus.

[0132] SEQ ID NO:3:MRLAVGALLVCAVLGLCLA

[0133] SEQ ID NO:4:MKWVTFISLLFSSAYS

[0134] The nucleotide sequence shown in SEQ ID NO:5 after codon optimization:

[0135]

[0136] The nucleotide sequence shown in SEQ ID NO:6 after codon optimization:

[0137]

[0138] The nucleotide sequence shown in SEQ ID NO:7 after codon optimization:

[0139]

[0140] Example 2: Construction of expression vector and expression in CHO cells

[0141] Plasmids were prepared in large quantities and transformed into DH5α competent cells (purchased from Tiangen Biotech). Single colonies were picked for identification. Bacterial cultures with correct sequencing results were transferred to 100 ml of LB medium and incubated overnight at 37°C. The bacterial culture was then collected, and plasmids were extracted according to the instructions of the plasmid extraction kit (purchased from Qiagen). Endotoxins were removed, and the concentration, A260 / 280 ratio, and endotoxin levels were measured. The endotoxin test was negative, and the bands were detected by electrophoresis.

[0142] CHO cells (purchased from Thermo) were transfected using the ExpiCHO transfection kit (Thermo Biotechnology) according to the manufacturer's instructions. Enhancers and adjuvants were added 15-24 hours later. Cells were cultured at 36.5℃, 95 rpm, and 8% CO2 for approximately 12 days. Once the cell viability was above 70%, the supernatant was collected by centrifugation at 3500 rpm for 30 minutes at 4℃.

[0143] Example 3: ELISA to identify antibody expression levels

[0144] Take an equal amount of supernatant containing plasmids of the above three signal peptides that have been transiently transfected into CHO cells for 12 days, add it to an ELISA plate and coat it overnight at 4°C.

[0145] After coating, the plates were washed three times with a plate washer and then sealed in a 37°C constant temperature incubator with skim milk powder for 1 hour.

[0146] Then, add the TRF antibody (purchased from Abcam) diluted with PBS and incubate at 37°C for 2 hours. Wash the plate 3 times after incubation. Add the secondary antibody (purchased from Sangon Biotech) and incubate at 37°C for 30 minutes. Wash 5 times, gently pat dry, add TMB chromogenic buffer, and develop at room temperature in the dark for 10 minutes until a light blue color is visible to the naked eye. Add 50 μL of stop solution per well and measure the OD value at 450 nm / 630 nm using a microplate reader.

[0147] The result is from Figure 1 As shown. According to Figure 1 The TRF expression vector linked to the Albumin signal peptide secreted more TRF protein in the supernatant of CHO cells than its own signal peptide and α-factor signal peptide.

[0148] Example 4: Purification of the expression product

[0149] Collect 100 ml of supernatant from CHO cells transiently transfected with the Albumin signal peptide via a TRF vector, and filter it using a 0.22 μm needle filter to obtain the filtered cell supernatant. After filtration, pass the supernatant through a Ni-column affinity chromatography. The protein eluted with 50 mM Tris-HCl, 50 mM NaCl, 500 mM imidazole, pH 7.0 is the target protein.

[0150] Electrophoresis image as shown Figure 2 As shown, the molecular weight is close to 84.2 KD, which is the same as the theoretical value.

[0151] The concentration was detected using the BCA method at 2 mg / ml, with a standard curve R² = 0.997. The total protein content was 10 mg, and the purity reached 90%. The expression of the target protein TRF in the CHO cell supernatant was calculated to be 90 mg / L.

[0152] Example 5: ELISA for antigenicity identification

[0153] Using a transferrin detection kit (purchased from Yunclone), follow the instructions to dilute the purified TRF and natural TRF (Seebio:EDD0087A) expressed in this example to the same concentration, add 0.2 ng / well, add developing solution and incubate for 20 min, add stop solution to terminate, and then measure the OD value at 450 nm / 630 nm on a microplate reader.

[0154] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention. SEQUENCE LISTING <110> Guangzhou Da An Gene Co., Ltd. <120> Human transferrin and its preparation method <130> P210722-1CNCNA9 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 679 <212> PRT <213> Artificial sequence <400> 1 Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu Ala 1 5 10 15 Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro Ser 20 25 30 Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp Cys 35 40 45 Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp Ala 50 55 60 Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro Val 65 70 75 80 Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr Tyr 85 90 95 Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln Leu 100 105 110 Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp 115 120 125 Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg Lys 130 135 140 Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala Pro 145 150 155 160 Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro Gly 165 170 175 Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala Phe 180 185 190 Lys Cys Leu Lys Asn Gly Ala Gly Asp Val Ala Phe Val Lys His Ser 195 200 205 Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr Glu 210 215 220 Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys Asp 225 230 235 240 Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala Arg Ser Met 245 250 255 Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln Glu 260 265 270 His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser Pro 275 280 285 His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Phe Lys 290 295 300 Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr Val 305 310 315 320 Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Gln Glu Ala Pro Thr 325 330 335 Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu Arg 340 345 350 Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu Cys 355 360 365 Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn Gly 370 375 380 Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala Gly 385 390 395 400 Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn Lys Ser Asp 405 410 415 Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val Val 420 425 430 Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys Lys 435 440 445 Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met 450 455 460 Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe Phe 465 470 475 480 Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys Lys 485 490 495 Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys Glu 500 505 510 Glu Gly Gly Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys Gly 515 520 525 Asp Val Ala Phe Val Lys His Gln Thr Val Pro Gln Asn Thr Gly Gly 530 535 540 Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr Glu 545 550 555 560 Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala Asn 565 570 575 Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys Asp 580 585 590 Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu Phe 595 600 605 Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg Ser 610 615 620 Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala Lys 625 630 635 640 Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr Val 645,650,655 Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu Glu 660 665 670 Ala Cys Thr Phe Arg Arg Pro 675 <210> 2 <211> 2037 <212> DNA <213> Artificial sequence <400> 2 gtgccagata agactgtgcg atggtgcgca gtgtccgaac atgaggccac caagtgccag 60 agcttcagag atcacatgaa gtctgtgatc cctagcgatg gcccctccgt ggcctgtgtc 120 aagaaagcct cttatctgga ctgcatccgg gccatcgccg ccaacgaggc cgatgccgtc 180 acactggatg ccggactggt gtacgacgcc tacctggccc ctaacaacct gaagcctgtg 240 gtggccgagt tctacggctc caaagaggac ccccagacct tctactacgc cgtggctgtc 300 gtgaagaagg actcgggctt ccagatgaac cagctgagag gcaagaagtc ctgccacacc 360 ggcctgggca gatctgctgg ctggaacatc cctatcgggc tgctgtactg cgacttgcct 420 gaaccccgga agcctctgga aaaggccgtg gccaacttct tctctggctc ttgtgctcca 480 tgtgccgacg gaacagactt ccctcagctg tgccagctgt gtcctggctg cggctgctcc 540 accctgaatc agtacttcgg ctactccgga gccttcaagt gcctgaaaaa cggggccggc 600 gatgtggcct ttgtgaagca ctccaccatc ttcgagaatc tggctaataa ggccgacaga 660 720 tgccatctgg caaagtgcc atctcacacc gtggttgctc ggtccatggg aggcaaagaa 780 gatctgatct gggagctgct gaaccaggct caagagcact tcggcaagga caaatccaag 840 gagttccagc tgttctcctc tcctcacggc aaagacttgc tgtttaagga ctccgcccat 900 ggcttcttca aggtgccacc tcggatggac gctaagatgt acctgggata cgagtacgtg 960 accgctatca gaaacctcag agagggcacc tgccaggaag cccctaccga cgagtgcaag 1020 cctgtcaagt ggtgcgccct gtcccaccac gagagactga agtgcgatga gtggtccgtg 1080 aactccgtgg gcaagatcga gtgtgtgtct gccgaaacca ccgaggactg cattgccaag 1140 atcatgaacg gcgaggctga cgccatgtcc ctggacggcg gcttcgtgta catcgctggc 1200 aaatgtggcc tggtccccgt gctggctgag aactacaaca agtctgataa ctgcgaggat 1260 acccctgaag cgggctactt cgccgtggct gtggtgaaga agtctgctag cgacctgacc 1320 tgggacaacc tgaagggcaa aaagtcctgt catacagctg tgggaagaac cgccggctgg 1380 aacatcccca tgggcctgct gtacaacaag atcaaccact gcagattcga cgagtttttc 1440 agcgagggct gtgcacctgg cagcaagaag gattcatctc tgtgcaagct gtgcatgggc 1500 tctggcctga acctctgcga gcctaacaac aaagaggaag gaggcggcta caccggcgct 1560 tttagatgcc tggtggaaaa gggcgacgtg gccttcgtga agcaccagac agtgcctcag 1620 aacaccggcg gaaagaaccc cgacccttgg gccaagaacc tgaacgagaa ggactacgag 1680 ctgctgtgtc tggatggcac acgcaagcct gtggaagaat acgctaactg tcacctagcc 1740 agagctccta atcacgctgt ggtgacccgg aaagacaagg aggcctgcgt gcacaagatc 1800 ctgcggcaac agcagcacct gtttggctcc aacgtgaccg actgttccgg caatttctgc 1860 ctgttcaggt ctgagacaaa ggatctgctg ttccgggacg acaccgtgtg cttagccaag 1920 ctgcacgaca gaaacaccta cgaaaagtac ctgggcgaag agtacgtgaa ggctgttggc 1980 aatctgagaa agtgctctac cagctccctc ctggaagctt gcacctttcg gcggccc 2037 <210> 3 <211> 19 <212> PRT <213> Artificial Sequence <400> 3 Met Arg Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu 1 5 10 15 Cys Leu Ala <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <400> 4 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Ser Ser Ala Tyr Ser 1 5 10 15 <210> 5 <211> 2094 <212> DNA <213> Artificial Sequence <400> 5 atgcgactag cagtgggtgc actactagtg tgtgccgtgc tgggcctgtg cctggctgtg 60 ccagataaga ctgtgcgatg gtgcgcagtg tccgaacatg aggccaccaa gtgccagagc 120 ttcagagatc acatgaagtc tgtgatccct agcgatggcc cctccgtggc ctgtgtcaag 180 aaagcctctt atctggactg catccgggcc atcgccgcca acgaggccga tgccgtcaca 240 ctggatgccg gactggtgta cgacgcctac ctggcccta acaacctgaa gcctgtggtg 300 gccgagttct acggctccaa agaggacccc cagaccttct actacgccgt ggctgtcgtg 360 aagaaggact cgggcttcca gatgaaccag ctgagaggca agaagtcctg ccacaccggc 420 ctgggcagat ctgctggctg gaacatccct atcgggctgc tgtactgcga cttgcctgaa 480 ccccggaagc ctctggaaaa ggccgtggcc aacttcttct ctggctcttg tgctccatgt 540 gccgacggaa cagacttccc tcagctgtgc cagctgtgtc ctggctgcgg ctgctccacc 600 ctgaatcagt acttcggcta ctccggagcc ttcaagtgcc tgaaaaacgg ggccggcgat 660 gtggccttg tgaagcactc caccatctttc gagaatctgg ctaataaggc cgacagagac 720 cagtatgaac tgctgtgcct ggacaacacc cggaagcctg tggacgaata caaggattgc 780 catctggcac aagtgccatc tcacaccgtg gttgctcggt ccatgggagg caaagaagat 840 ctgatctggg agctgctgaa ccaggctcaa gagcacttcg gcaaggacaa atccaaggag 900 ttccagctgt tctcctcc tcacggcaaa gacttgctgt ttaaggactc cgcccatggc 960 ttcttcaagg tgccacctcg gatggacgct aagatgtacc tgggatacga gtacgtgacc 1020 gctatcagaa acctcagaga gggcacctgc caggaagccc ctaccgacga gtgcaagcct 1080 gtcaagtggt gcgccctgtc ccaccacgag agactgaagt gcgatgagtg gtccgtgaac 1140 tccgtgggca agatcgagtg tgtgtctgcc gaaaccaccg aggactgcat tgccaagatc 1200 atgaacggcg aggctgacgc catgtccctg gacggcggct tcgtgtacat cgctggcaaa 1260 tgtggcctgg tccccgtgct ggctggagaac tacaacaagt ctgataactg cgaggatacc 1320 cctgaagcgg gctacttcgc cgtggctgtg gtgaagaagt ctgctagcga cctgacctgg 1380 gacaacctga agggcaaaaa gtcctgtcat acagctgtgg gaagaaccgc cggctggaac 1440 atcccatgg gcctgctgta caacaagatc aaccactgca gattcgacga gtttttcagc 1500 gagggctgtg cacctggcag caagaaggat tcatctctgt gcaagctgtg catgggctct 1560 ggcctgaacc tctgcgagcc taaaaaaa gagaggag gcggctacac cggcgctttt 1620 agatgcctgg tggaaaaggg cgacgtggcc ttcgtgaagc accagacagt gcctcagaac 1680 accggcggaa agaaccccga cccttgggcc aagaacctga acgagaagga ctacgagctg 1740 ctgtgtctgg atggcacacg caagcctgtg gaagaatacg ctaactgtca cctagccaga 1800 gctcctaatc acgctgtggt gacccggaaa gacaaggagg cctgcgtgca caagatcctg 1860 cggcaacagc agcacctgtt tgctccaac gtgaccgact gttccggcaa tttctgcctg 1920 ttcaggtctg agacaaagga tctgctgttc cgggacgaca ccgtgtgctt agccaagctg 1980 cacgacagaa acacctacga aaagtacctg ggcgaagagt acgtgaaggc tgttggcaat 2040 ctgagaaagt gctctaccag ctccctcctg gaagcttgca cctttcggcg gcc 2094 <210> 6 <211> 2085 <212> DNA <213> Artificial sequence <400> 6 atgaagtggg tgacttttat cagtctacta tttagctctg cctactccgt gccagataag 60 actgtgcgat ggtgcgcagt gtccgaacat gaggccacca agtgccagag cttcagagat 120 cacatgaagt ctgtgatccc tagcgatggc ccctccgtgg cctgtgtcaa gaaagcctct 180 tatctggact gcatccgggc catcgccgcc aacgaggccg atgccgtcac actggatgcc 240 ggactggtgt acgacgccta cctggcccct aacaacctga agcctgtggt ggccgagttc 300 tacggctcca aagaggaccc ccagaccttc tactacgccg tggctgtcgt gaagaaggac 360 tcgggcttcc agatgaacca gctgagaggc aagaagtcct gccacaccgg cctgggcaga 420 tctgctggct ggaacatccc tatcgggctg ctgtactgcg acttgcctga accccggaag 480 cctctggaaa aggccgtggc caacttcttc tctggctctt gtgctccatg tgccgacgga 540 acagacttcc ctcagctgtg ccagctgtgt cctggctgcg gctgctccac cctgaatcag 600 tacttcggct actccggagc cttcaagtgc ctgaaaaacg gggccggcga tgtggccttt 660 gtgaagcact ccaccatctt cgagaatctg gctaataagg ccgacagaga ccagtatgaa 720 ctgctgtgcc tggacaacac ccggaagcct gtggacgaat acaaggattg ccatctggca 780 caagtgccat ctcacaccgt ggttgctcgg tccatgggag gcaaagaaga tctgatctgg 840 gagctgctga accaggctca agagcacttc ggcaaggaca aatccaagga gttccagctg 900 ttctcctctc ctcacggcaa agacttgctg tttaaggact ccgcccatgg cttcttcaag 960 gtgccacctc ggatggacgc taagatgtac ctgggatacg agtacgtgac cgctatcaga 1020 aacctcagag agggcacctg ccaggaagcc cctaccgacg agtgcaagcc tgtcaagtgg 1080 tgcgccctgt cccaccacga gagactgaag tgcgatgagt ggtccgtgaa ctccgtgggc 1140 aagatcgagt gtgtgtctgc cgaaaccacc gaggactgca ttgccaagat catgaacggc 1200 gaggctgacg ccatgtccct ggacggcggc ttcgtgtaca tcgctggcaa atgtggcctg 1260 gtccccgtgc tggctgagaa ctacaacaag tctgataact gcgaggatac ccctgaagcg 1320 ggctacttcg ccgtggctgt ggtgaagaag tctgctagcg acctgacctg ggacaacctg 1380 aagggcaaaa agtcctgtca tacagctgtg ggaagaaccg ccggctggaa catccccatg 1440 ggcctgctgt acaacaagat caaccactgc agattcgacg agtttttcag cgagggctgt 1500 gcacctggca gcaagaagga ttcatctctg tgcaagctgt gcatgggctc tggcctgaac 1560 ctctgcgagc ctaacaacaa agaggaagga ggcggctaca ccggcgcttt tagatgcctg 1620 gtggaaaagg gcgacgtggc cttcgtgaag caccagacag tgcctcagaa caccggcgga 1680 aagaaccccg acccttgggc caagaacctg aacgagaagg actacgagct gctgtgtctg 1740 gatggcacac gcaagcctgt ggaagaatac gctaactgtc acctagccag agctcctaat 1800 cacgctgtgg tgacccggaa agacaaggag gcctgcgtgc acaagatcct gcggcaacag 1860 cagcacctgt ttggctccaa cgtgaccgac tgttccggca atttctgcct gttcaggtct 1920 gagacaaagg atctgctgtt ccgggacgac accgtgtgct tagccaagct gcacgacaga 1980 aacacctacg aaaagtacct gggcgaagag tacgtgaagg ctgttggcaa tctgagaaag 2040 tgctctacca gctccctcct ggaagcttgc acctttcggc ggccc 2085 <210> 7 <211> 2304 <212> DNA <213> Artificial sequence <400> 7 atgcgatttc caagtatctt tactgcagtg ctgtttgctg cttcttctgc tctggccgct 60 cctgtcaata ccacaaccga ggacgagacc gcccagatcc ccgccgaggc tgtgatcgga 120 tacagcgatc tcgagggcga cttcgacgtg gccgtgctgc cttctccaa ctccaccaac 180 aacggcctgc tgttcatcaa caccaccatc gcctctatcg ccgccaagga ggaaggcgtg 240 tcctggaaa agagagaggc cgaagctgtg ccagataaga ctgtgcgatg gtgcgcagtg 300 tccgaacatg aggccaccaa gtgccagagc ttcagagatc acatgaagtc tgtgatccct 360 agcgatggcc cctccgtggc ctgtgtcaag aaagcctctt atctggactg catccgggcc 420 atcgccgcca acgaggccga tgccgtcaca ctggatgccg gactggtgta cgacgcctac 480 ctggccccta acaacctgaa gcctgtggtg gccgagttct acggctccaa agaggacccc 540 cagaccttct actacgccgt ggctgtcgtg aagaaggact cgggcttcca gatgaaccag 600 ctgagaggca agaagtcctg ccacaccggc ctgggcagat ctgctggctg gaacatccct 660 atcgggctgc tgtactgcga cttgcctgaa ccccggaagc ctctggaaaa ggccgtggcc 720 aacttcttct ctggctcttg tgctccatgt gccgacggaa cagacttccc tcagctgtgc 780 cagctgtgtc ctggctgcgg ctgctccacc ctgaatcagt acttcggcta ctccggagcc 840 ttcaagtgcc tgaaaaacgg ggccggcgat gtggcctttg tgaagcactc caccatcttc 900 gagaatctgg ctataaggc cgacagagac cagatatgaac tgctgtgcct ggacaacacc 960 cggaagcctg tggacgaata caaggattgc catctggcac aagtgccatc tcacaccgtg 1020 gttgctcggt ccatgggagg caaagagat ctgatctggg agctctctgaa ccaggctcaa 1080 gagcacttcg ggaggacaa atccaaggag ttccagctgt tctcctctcc tcacggcaaa 1140 gacttgctgt ttaaggactc cgcccatggc ttcttcaagg tgccacctcg gatggacgct 1200 aagatgtacc tgggatacga gtacgtgacc gctatcagaa acctcagaga gggcacctgc 1260 caggaagccc ctaccgacga gtgcaagcct gtcaagtggt gcgccctgtc ccaccacgag 1320 agactgaagt gcgatgagtg gtccgtgaac tccgtgggca agatcgagtg tgtgtctgcc 1380 gaaaccaccg aggactgcat tgccaagatc atgaacggcg aggctgacgc catgtccctg 1440 gacggcggct tcgtgtacat cgctggcaaa tgtggcctgg tccccgtgct ggctggagaac 1500 tacaacaagt ctgataactg cgaggatacc cctgaagcgg gctacttcgc cgtggctgtg 1560 1620 acagctgtgg gaagaaccgc cggctggaac atccccatgg gcctgctgta caacaagatc 1680 1740 tcatctctgt gcaagctgtg catgggctct ggcctgaacc tctgcgagcc taaaaaaa 1800 ggagaggag gcggctacac cggcgctttt agatgcctgg tggaaaaggg cgacgtggcc 1860 ttcgtgaagc accagacagt gcctcagaac accggcggaa agaacccccga cccttgggcc 1920 1980 gaagaatacg ctaactgtca cctagccaga gctcctaatc acgctgtggt gacccggaaa 2040 gaagaggagg cctgcgtgca gaagatcctg cggcaacagc agcacctgtt tggctccaac 2100 gtgaccgact gttccggcaa tttctgcctg ttcaggtctg agaaaagga tctgctgttc 2160 cgggacgaca ccgtgtgctt agccaagctg cacgacagaa acacctacga aaagtacctg 2220 ggcgaagagt acgtgaaggc tgttggcaat ctgagaaagt gctctaccag ctccctcctg 2280 gaagcttgca cctttcggcg gccc 2304

Claims

1. A codon-optimized polynucleotide, characterized in that, The polynucleotide is a nucleotide sequence as shown in SEQ ID NO.

6.

2. A fusion protein, characterized in that, The amino acid sequence of the fusion protein is identical to the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.

6.

3. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 1.

4. A host cell, characterized in that, The host cell contains the expression vector as described in claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is a CHO cell.

6. A method for preparing a fusion protein, characterized in that, The method includes the following steps: Under suitable expression conditions, the host cells as described in claim 4 or 5 are cultured to express the target protein; and The target protein was isolated.

7. A reagent kit, characterized in that, The kit contains the fusion protein of claim 2, the polynucleotide of claim 1, the expression vector of claim 3, or the host cell of claim 4 or 5.

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