SC1-70 antigen truncation body, its preparation method and application

Abstract

This application discloses a truncated form of the SC1-70 antigen, its preparation method, and application. This application develops a genetic engineering-based method for preparing the truncated form of the SC1-70 antigen, achieving efficient and stable soluble expression in an Escherichia coli expression system. The resulting truncated form of the SC1-70 antigen exhibits activity comparable to that of the SC1-70 antigen.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to an SC1-70 antigen truncation, a preparation method and application thereof. Background Art

[0002] Sc1-70 is a 70 kDa protein derived from the C-terminal fragment of DNA topoisomerase I (TOP1), named for its 70 kDa antigenic molecular weight. DNA topoisomerase I has a native molecular weight of 100 kDa and is located in the nucleoplasm and nucleolus, with the highest concentration in the nucleolus. It plays a role in loosening localized helices during DNA replication and transcription. Sc1-70 antibodies belong to the category of anti-extractable nuclear antigen antibodies, recognizing DNA topoisomerase I. These antibodies are found in various autoimmune diseases and are relatively specific for systemic sclerosis. Therefore, detecting Sc1-70 antibodies can improve the diagnostic rate of systemic sclerosis, facilitating differential diagnosis and enhancing treatment efficacy.

[0003] There are currently a variety of clinical methods for detecting Sc1-70 antibodies, such as indirect immunofluorescence, immune double diffusion, enzyme-linked immunosorbent assay, etc. These methods all require the use of Sc1-70 protein. In the prior art, the common methods for obtaining Sc1-70 protein are extraction and recombinant protein methods. The purification and extraction of natural Sc1-70 protein has complicated processes, low yield and low purity, which cannot meet the growing demand for detection; the recombinant Sc1-70 protein method based on genetic engineering, although it overcomes the defect of raw material shortage of the extraction method, the prepared protein is expressed in the form of inclusion bodies, with extremely low yield, low activity of the obtained protein and difficulty in purification, which cannot meet production needs.

[0004] Therefore, there is still a need in the art to develop a method for preparing Sc1-70 protein with high yield, high protein activity and easy purification. Summary of the Invention

[0005] The object of the present invention is to provide a truncated form of the SC1-70 antigen.

[0006] The purpose of the present invention is to provide a method for preparing a recombinant SC1-70 antigen truncate.

[0007] Another object of the present invention is to provide a polynucleotide sequence encoding a truncated form of the SC1-70 antigen.

[0008] Another object of the present invention is to provide a vector compatible with the polynucleotide sequence encoding the truncated form of the SC1-70 antigen.

[0009] Another object of the present invention is to provide a kit containing a polynucleotide sequence encoding a truncated form of the SC1-70 antigen.

[0010] In order to solve the above technical problems, the first aspect of the present invention provides a truncated form of the SC1-70 antigen, wherein the truncated form of the SC1-70 antigen is selected from any one of the following:

[0011] (i) a polypeptide having a sequence as shown in SEQ ID NO. 2; and

[0012] (ii) A polypeptide having a homology greater than 95% with SEQ ID NO. 2.

[0013] In a second aspect of the present invention, a polynucleotide encoding a truncated form of the SC1-70 antigen is provided, wherein the polynucleotide is codon-optimized and selected from any one of the following:

[0014] (i) a polynucleotide having the sequence shown in SEQ ID NO. 3;

[0015] (ii) a polynucleotide having a homology greater than 95% with the sequence shown in SEQ ID NO. 3; and

[0016] (iii) a polynucleotide having a sequence complementary to the polynucleotide described in (i) or (ii).

[0017] The third aspect of the present invention provides an expression vector, which includes the polynucleotide provided by the first aspect of the present invention.

[0018] In some preferred embodiments, the expression vector comprises a polynucleotide sequence expressing a His×6 tag. More preferably, in the expression vector, the 5' end of the polynucleotide is connected to a polynucleotide sequence expressing a His×6 tag.

[0019] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

[0020] In a fourth aspect, the present invention provides a host cell, wherein the host cell comprises the expression vector provided in the second aspect of the present invention; or

[0021] The polynucleotide provided in the first aspect of the present invention is integrated into the genome of the host cell.

[0022] In some preferred embodiments, the host cell is Escherichia coli.

[0023] In some preferred embodiments, the host cell is Escherichia coli Rosetta (DE3) strain.

[0024] The fifth aspect of the present invention provides a method for preparing a truncated form of the SC1-70 antigen, the method comprising the steps of: culturing the host cell according to the third aspect of the present invention to express a target protein; and

[0025] Separating the target protein to obtain the SC1-70 antigen truncation;

[0026] Wherein, the target protein has an amino acid sequence such as the amino acid sequence shown in SEQ ID NO: 1.

[0027] In some preferred embodiments, the host cell is obtained by transforming Escherichia coli with a plasmid containing the polynucleotide described in the first aspect of the present invention.

[0028] In some preferred embodiments, the host cells are cultured in SB, TB or SOC medium. To obtain a large amount of soluble expression, in a more preferred embodiment, the host cells are cultured in SB medium.

[0029] In some preferred embodiments, the host cells are cultured in a shaking environment.

[0030] In some preferred embodiments, when culturing the host cell, the culture medium used contains a kanamycin resistance gene.

[0031] In some preferred embodiments, when culturing the host cells, IPTG is used for induction to express the target protein.

[0032] In some preferred embodiments, when culturing the host cells, the cells are cultured until the OD600 is between 0.6 and 0.8, and then induced with IPTG to express the target protein.

[0033] In some preferred embodiments, the step of separating the target protein comprises:

[0034] The supernatant of the crushed target protein is passed through a chromatography column for elution, and the eluate is collected.

[0035] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column, such as HisTrap™ FF.

[0036] The sixth aspect of the present invention provides a kit, which comprises: the polynucleotide provided in the first aspect of the present invention; or

[0037] The expression vector provided in the second aspect of the present invention; or

[0038] The host cell according to the third aspect of the present invention; or

[0039] Or the SC1-70 antigen truncation prepared by the method according to the fourth aspect of the present invention.

[0040] Compared with the prior art, the present invention has at least the following advantages:

[0041] (1) The present invention develops a method for preparing SC1-70 antigen truncations based on genetic engineering, achieving efficient and stable soluble expression in an E. coli expression system, and the activity of the obtained SC1-70 antigen truncations is equivalent to that of the SC1-70 antigen;

[0042] (2) In a preferred embodiment of the present invention, the N-terminus carries a polynucleotide sequence encoding a (His)6 tag, which further improves the amount of soluble expression produced by the system, and no longer requires steps such as tag removal and secondary purification. A high-purity, high-yield target protein is obtained in one step, which simplifies the purification steps, improves the recovery rate and purity, and can obtain a highly active recombinant Sc1-70 protein, providing a diagnostic reagent raw material for the clinical differential diagnosis of systemic scleroderma and laying the foundation for the development of a rapid detection kit for Sc1-70 antibodies;

[0043] (3) In a preferred embodiment of the present invention, the soluble expression of the target protein is further increased by optimizing the host cell culture method, such as using optimized induction conditions, optimizing the culture medium and culture temperature.

[0044] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail 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 listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] One or more embodiments are exemplarily described by the figures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments.

[0046] Figure 1 This is a diagram showing the SDS-PAGE identification results of the SC1-70 antigen according to an embodiment of the present invention;

[0047] Figure 2 1 is an electrophoresis diagram of SC1-70 antigen according to an embodiment of the present invention. DETAILED DESCRIPTION

[0048] Although a recombinant SC1-70 protein expression system based on genetic engineering has been developed in the prior art, the yield is low, the product stability is poor, the proportion of inclusion bodies in the product is large, and the proportion of soluble protein is too small to be purified. The inventors unexpectedly discovered in their research that the SC1-70 protein truncation not only has activity comparable to that of the SC1-70 protein, but also when prepared using the recombinant protein method, the proportion of soluble protein in the resulting product is significantly increased. Based on this, the inventors developed an expression system for the SC1-70 protein truncation, and further optimized the synonymous codon preference to obtain a polynucleotide sequence encoding the SC1-70 truncation that can express the target protein in large quantities in the Escherichia coli expression system. The expressed soluble target protein has a high yield, high activity, and good stability.

[0049] In a more preferred embodiment of the present invention, the inventors added a polynucleotide sequence encoding a (His)6 tag to the N-terminus of the polynucleotide sequence encoding the SC1-70 antigen after optimization of synonymous codon preference, thereby further increasing the amount of soluble expression.

[0050] In a more preferred embodiment of the present invention, the inventors optimized the host cell culture method by using kanamycin-resistant SB medium combined with IPTG-induced culture, thereby relatively increasing the soluble expression level of the target protein.

[0051] Obtain target gene / target protein related nucleic acid sequence

[0052] The full-length nucleotide sequence or fragments of the target protein or its components in the present invention can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, particularly open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified in the correct order.

[0053] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0054] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0055] Methods using PCR technology to amplify DNA / RNA are preferably used to obtain the genes 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. Amplified DNA / RNA fragments can be separated and purified using conventional methods, such as by gel electrophoresis.

[0056] In one embodiment of the present invention, the amino acid sequence of the target protein (SEQ ID NO: 1) is analyzed by the NCBI database, and then truncated to obtain the 201-765aa target protein truncated sequence (SEQ ID NO: 3) to obtain the target gene sequence information.

[0057] Synonymous codon preference optimization

[0058] To overcome the potential problem of reduced yield when expressing heterologous proteins in E. coli, the present invention relates to polynucleotide sequences optimized for synonymous codon preference. The obtained 201-765aa target gene sequence was subjected to synonymous codon preference optimization. This optimized target gene sequence (SEQ ID NO: 4) can express the same amino acid sequence as the target protein, while improving the stability and efficiency of the expression process, ultimately maintaining a high activity of the target protein.

[0059] The present invention also relates to a polynucleotide having a homology greater than 95% with the sequence shown in SEQ ID NO: 4; and a polynucleotide complementary to the sequence shown in SEQ ID NO: 3.

[0060] The present invention also relates to a polynucleotide having a homology greater than 95% with the sequence shown in SEQ ID NO: 4; and a polynucleotide complementary to the sequence shown in SEQ ID NO: 3.

[0061] Target gene vector

[0062] The present invention also relates to vectors comprising the polynucleotides of the present invention. As used herein, "vector" refers to a linear or circular DNA molecule comprising a segment encoding a protein of interest operably linked to other segments that provide for its transcription. Such additional segments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, vectors, and the like. The vector segment may be derived from a host organism, another organism, a plasmid, or viral DNA, or may be synthetic. The vector may be synthetic or any expression vector that can be readily subjected to recombinant DNA procedures, and the choice of vector generally depends on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be a vector that, when introduced into a host cell, is integrated into the host cell genome and replicates along with the chromosome into which it is integrated. In one embodiment, the vector of the present invention is an expression vector. In one embodiment of the present invention, pET-28a(+) was selected as the vector to achieve more efficient expression efficiency.

[0063] 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 the present invention and appropriate 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 effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Illustratively, a DNA endonuclease is used to cut the vector DNA molecule into a linear molecule that can be connected to the exogenous gene, and then the codon-optimized target gene fragment is connected to the vector. The insertion of the exogenous DNA fragment can be achieved by selectively ligating the exogenous DNA fragment with a single restriction endonuclease, directional cloning of double restriction endonuclease fragments, ligating the exogenous DNA fragment with different restriction endonuclease sites, blunt end ligation, artificial linker ligation, or oligonucleotide end ligation.

[0064] In one embodiment of the present invention, the vector further comprises a polynucleotide sequence expressing a His×6 tag. Preferably, the polynucleotide sequence expressing a His×6 tag is connected to the 5' end (N-terminus) of the target gene sequence, thereby improving the soluble expression of the target gene and facilitating subsequent separation and purification.

[0065] The vector containing the target gene is transformed into the host cell

[0066] The present invention also relates to host cells produced by genetic engineering using the vectors or fusion protein coding sequences of the present invention. A vector containing a codon-optimized target gene can be inserted, transfected, or otherwise transformed into a host cell by known methods, thereby obtaining a transformant containing the codon-optimized target gene of the present invention and capable of expressing the target protein. In the present invention, a "host cell" is a cell into which an exogenous polynucleotide and / or vector has been introduced. The host cell can be a eukaryotic host cell or a prokaryotic host cell. The host cell is preferably a bacterium, and is preferably Escherichia coli, more preferably Escherichia coli Rosetta (DE3) strain.

[0067] Method for preparing target protein

[0068] The present invention also relates to a method for preparing a target protein, which can be expressed or produced using the polynucleotide sequence of the present invention. Generally, the following steps are involved:

[0069] (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0070] (2) host cells cultured in a suitable culture medium;

[0071] (3) Isolate and purify proteins from culture medium or cells.

[0072] Among them, in step (1), the transformation or transduction of a suitable host cell with the recombinant expression vector containing the polynucleotide can be carried out by conventional techniques well known to those skilled in the art. When the host is Escherichia coli, heat shock method and electroporation method can be used.

[0073] The transformant obtained can be cultivated with conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the cultivation can be selected from various conventional culture media, preferably SB, TB or SOC culture medium. Cultivate under conditions suitable for host cell growth. After the host cells grow to a suitable cell density, induce the promoter of selection with a suitable method (such as temperature conversion or chemical induction), and the cells are cultivated for a period of time. In order to promote the expression of the target protein and to improve the expression of the soluble protein, a preferred embodiment of the present invention uses the host cells cultivated with SB culture medium, and the culture medium used contains the kanamycin resistance gene.

[0074] The protein in the above method can be expressed in the cell or on the cell membrane or secreted outside the cell. If necessary, its physical, chemical and other characteristics can be utilized to separate and purify the protein by various separation methods. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, infiltration sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and the combination of these methods. In one embodiment of the invention, affinity chromatography molecular target protein is used.

[0075] In the present invention, any exemplary or exemplary wording (e.g., "") provided for certain embodiments herein is used only to better present the present invention and does not limit the scope of the present invention claimed in other ways. Any wording herein should not be interpreted as indicating an element not described in the claims that is indispensable for the implementation of the present invention.

[0076] If a definition or use of a term in a referenced document is inconsistent or inconsistent with the definition of that term as described herein, the definition of that term as described herein applies and the definition of that term in the referenced document does not apply.

[0077] Various terms are used herein as follows. If a term used in a claim is not defined below, it should be given the broadest definition persons in the art have given that term as reflected in printed publications or issued patents at the time of filing.

[0078] As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., nucleic acid or polypeptide) present in its natural source. In one embodiment, the nucleic acid or polypeptide is found only in the presence of solvents, buffers, ions, or other components that are normally present in a solution thereof, if any. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.

[0079] As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0080] The present invention also relates to variants of the aforementioned polynucleotides, which encode protein fragments, analogs, and derivatives having the same amino acid sequence as the present invention. These polynucleotide variants may 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 an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded polypeptide.

[0081] As used herein, the term "codon optimization" refers to a method for improving gene synthesis efficiency by avoiding the use of low-utilization or rare codons based on the differences in codon usage exhibited by organisms actually expressing or producing proteins (including Escherichia coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

[0082] As used herein, the terms "homology" and "identity" are used interchangeably and refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be measured by the following method. The nucleotide or amino acid sequence of a polynucleotide or polypeptide is arranged, and the number of positions containing identical nucleotides or amino acid residues in the arranged polynucleotide or polypeptide is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the arranged polynucleotide or polypeptide. A polynucleotide can be different in one position, for example, according to comprising different nucleotides (i.e., replacement or variation) or deletions of nucleotides (i.e., insertion or deletion of one or two nucleotides in a polynucleotide). A polypeptide can be different in one position, for example, by containing an amino acid (i.e., replacement or variation) or deletion of an amino acid (i.e., insertion of an amino acid or amino acid deletion in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of amino acid residues in a polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0083] As used herein, the terms "sequence complement" and "reverse sequence complement" are used interchangeably to refer to a sequence that is in the opposite direction of the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complement sequence is GTTCAT.

[0084] As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. After expression, the host cells or expression products may be harvested, i.e., recovered.

[0085] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0086] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0087] Example 1: Construction of SC1-70 plasmid and transfection of host cells

[0088] (1) The amino acid sequence of human TOP1 protein was obtained as shown in SEQ ID NO: 1, and 201-765aa was truncated (shown in SEQ ID NO: 3). The gene sequence was analyzed and obtained, and the synonymous codon preference was optimized. After the synonymous codon preference optimization of Escherichia coli, SEQ ID NO. 2 (encoding the complete TOP1 protein), SEQ ID NO: 4 (encoding the 201-765aa truncation), SEQ ID NO: 5 (encoding the 201-755aa truncation), and SEQ ID NO: 6 (encoding the 188-765aa truncation) were obtained. The vectors were respectively connected to pET-28a (+), and the N-terminal fusion expression (His) 6 tag was expressed to synthesize recombinant expression plasmids.

[0089] (2) Introduction of recombinant plasmid into host Escherichia coli

[0090] Take 1 μL of the expression plasmid prepared in step (1) and add it to 30 μL of competent E. coli Rosetta (DE3) in an ice bath. Place it on ice for 20 minutes, heat shock it for 90 seconds, and immediately place it on ice for 2 minutes. Add 400 μL of SOC medium without antibiotics and culture it at 37°C with shaking at 220 rpm for 50 minutes. Take 100 μL of the bacterial solution and evenly spread it on an LB plate containing 100 μg / mL kanamycin resistance and culture it in a 37°C incubator overnight.

[0091] The amino acid sequence of human TOP1 protein is SEQ ID NO: 1:

[0092] MSGDHLHNDSQIEADFRLNDSHKHKDKHKDREHRHKEHKKEKDREKSKHSNSEHKDSEKKHKEKEKTKHKDGSSEKHKDK

[0093] HKDRDKEKRKEEKVRASGDAKIKKEKENGFSSPPQIKDEPEDDGYFVPPKEDIKPLKRPRDEDDADYKPKKIKTEDTKKE

[0094] KKRKLEEEDGKLKKPKNKDKDKKVPEPDNKKKKPKKEEEQKWKWWEEERYPEGIKWKFLEHKGPVFAPPYEPLPENVKF

[0095] YYDGKVMKLSPKAEEVATFFAKMLDHEYTTKEIFRKNFFKDWRKEMTNEEKNIITNLSKCDFTQMSQYFKAQTEARKQMS

[0096] KEEKLKIKEENEKLLKEYGFCIMDNHKERIANFKIEPPGLFRGRGNHPKMGMLKRRIMPEDIIINCSKDAKVPSPPPGHK

[0097] WKEVRHDNKVTWLVSWTENIQGSIKYYIMLNPSSRIKGEKDWQKYETARRLKKCVDKIRNQYREDWKSKEMKVRQRAVALY

[0098] FIDKLALRAGNEKEEGETATDTVGCCSLRVEHINLHPELDGQEYVVEFDFLGKDSIRYYNKVPVEKRVFKNLQLFMENKQP

[0099] EDDLFDRLNTGILNKHLQDLMEGLTAKVFRTYNASITLQQQQLKELTAPDENIPAKILSYNRANRAVAILCNHQRAPPKTF

[0100] EKSMMNLQTKIDAKKEQLADARRDLKSAKADAKVMKDAKTKKVVESKKKAVQRLEEQLMKLEVQATDREENKQIALGTSK

[0101] LNYLDPRITVAWCKKWGVPIEKIYNKTQREKFAWAIDMADEDYEF;

[0102] Codon-optimized polynucleotide sequence encoding human TOP1 protein SEQ ID NO:2:

[0103] ATGAGTGGAGATCATCTACACAATGACTCACAAATTGAAGCGGACTTCCGTCTGAATGATAGCCATAAACATAAGGACAA

[0104] ACACAAAGACCGCGAACACCGCCATAAGGAACACAAGAAAGAGAAGGACCGTGAAAAGTCTAAGCACTCTAATAGCGAGC

[0105] ACAAGGATTCCGAGAAAAAGCACAAAGAAAAGGAAAAAACCAAACACAAGGACGGCAGCAGTGAAAAGCACAAGGACAAG

[0106] CATAAAGACCGCGATAAAGAGAAGCGAAAGGAGGAGAAGGTGCGCGCTTCCGGTGATGCGAAAATCAAGAAAGAAAAGGA

[0107] GAACGGCTTCAGCAGCCCGCCGCAGATTAAGGACGAACCGGAAGACGACGGCTATTTCGTCCCGCCGAAAGAGGACATCA

[0108] AACCGCTGAAACGTCCGCGTGATGAAGACGATGCGGACTACAAACCGAAGAAAATTAAGACCGAGGACACCAAAAAAGAA

[0109] AAGAAGCGTAAACTGGAGGAAGAGGAGGACGGTAAACTTAAGAAACCGAAAAACAAAGACAAAGACAAAAAAGTCCCGGA

[0110] GCCGGATAACAAAAAGAAGAAACCGAAGAAAGAGGAAGAACAGAAATGGAAATGGTGGGAAGAGGAACGTTATCCGGAGG

[0111] GCATTAAGTGGAAATTTTTGGAGCATAAGGGTCCAGTTTTCGCACCGCCGTATGAGCCTCTGCCGGAAAACGTTAAATTC

[0112] TATTACGATGGCAAAGTCATGAAGCTCTCACCGAAAGCGGAAGAGGTGGCCACCTTTTTCGCAAAAATGCTGGATCATGA

[0113] GTACACCACGAAGGAAATTTTTCGTAAAAACTTTTTTAAAGACTGGCGTAAAGAGATGACCAACGAAGAGAAGAACATCA

[0114] TCACCAACTTATCGAAATGCGATTTCACCCAAATGTCCCAGTACTTCAAAGCCCAGACCGAAGCACGTAAGCAGATGAGC

[0115] AAAGAGGAAAAATTGAAAATTAAAGAGGAGAACGAGAAGTTGCTGAAGGAATACGGCTTCTGCATTATGGATAATCATAA

[0116] GGAGCGCATTGCCAATTTCAAAATCGAACCGCCCGGTTTGTTCCGCGGGCGTGGCAATCATCCGAAGATGGGTATGCTGA

[0117] AGCGTAGAATCATGCCGGAGGACATTATCATCAACTGCAGCAAAGATGCGAAGGTTCCTAGCCCCCCGCCGGGTCATAAG

[0118] TGGAAAGAAGTTCGTCACGATAACAAGGTGACCTGGCTCGTTAGCTGGACCGAAAACATTCAGGGTAGCATCAAATACAT

[0119] CATGTTGAATCCGAGCTCTCGTATCAAGGGCGAAAAAGATTGGCAGAAGTACGAGACTGCTCGTCGCCTGAAAAAGTGCG

[0120] TGGATAAGATTCGTAATCAGTACCGCGAAGACTGGAAAAGCAAGGAGATGAAGGTTCGTCAGAGAGCGGTTGCCCTGTAT

[0121] TTTATCGATAAACTGGCGCTGCGAGCCGGTAATGAAAAAGAGGAGGGTGAAACCGCGGATACGGTGGGTTGTTGTAGCCT

[0122] GCGTGTTGAGCACATCAATCTGCACCCGGAGCTGGATGGCCAAGAGTACGTTGTCGAGTTCGATTTTTTGGGCAAGGACT

[0123] CGATCCGCTACTACAATAAGGTGCCAGTTGAAAAGCGCGTGTTCAAAAACCTGCAGCTTTTTATGGAAAACAAACAGCCG

[0124] GAGGACGACCTGTTCGATCGTCTGAACACCGGCATTCTGAACAAGCACCTGCAAGATCTGATGGAAGGTCTGACGGCTAA

[0125] AGTGTTTAGAACATATAATGCAAGCATTACTCTGCAACAACAGCTGAAGGAACTCACTGCGCCAGATGAAAACATCCCGG

[0126] CTAAGATCTTATCCTATAACCGTGCGAACCGCGCGGTGGCGATTCTGTGCAACCACCAGCGCGCTCCGCCGAAGACCTTT

[0127] GAGAAGTCCATGATGAATCTACAGACCAAGATCGACGCGAAGAAGGAGCAATTGGCGGACGCTAGACGTGATTTGAAGTC

[0128] TGCAAAGGCAGACGCTAAAGTGATGAAAGATGCGAAGACGAAAAAAGTGGTCGAAAGTAAGAAGAAAGCGGTGCAACGTC

[0129] TTGAGGAGCAACTGATGAAATTGGAAGTACAGGCGACCGATCGTGAGGAGAATAAACAAATTGCACTGGGTACCTCCAAG

[0130] CTGAACTATCTGGACCCGCGTATTACCGTAGCCTGGTGTAAAAAATGGGGTGTTCCGATCGAAAAGATCTACAACAAAAC

[0131] GCAACGCGAGAAGTTTGCCTGGGCAATTGACATGGCTGACGAGGATTATGAATTT

[0132] Amino acid sequence of the truncated TOP1 protein 201 - 765aa, SEQ ID NO:3:

[0133] QKWKWWEEERYPEGIKWKFLEHKGPVFAPPYEPLPENVKFYYDGKVMKLSPKAEEVATFFAKMLDHEYTTKEIFRKNFFK

[0134] DWRKEMTNEEKNIITNLSKCDFTQMSQYFKAQTEARKQMSKEEKLKIKEENEKLLKEYGFCIMDNHKERIANFKIEPPGL

[0135] FRGRGNHPKMGMLKRRIMPEDIIINCSKDAKVPSPPPGHKWKEVRHDNKVTWLVSWTENIQGSIKYIMLNPSSRIKGEKD

[0136] WQKYETARRLKKCVDKIRNQYREDWKSKEMKVRQRAVALYFIDKLALRAGNEKEEGETADTVGCCSLRVEHINLHPELDG

[0137] QEYVVEFDFLGKDSIRYYNKVPVEKRVFKNLQLFMENKQPEDDLFDRLNTGILNKHLQDLMEGLTAKVFRTYNASITLQQ

[0138] QLKELTAPDENIPAKILSYNRANRAVAILCNHQRAPPKTFEKSMMNLQTKIDAKKEQLADARRDLKSAKADAKVMKDAKT

[0139] KKVVESKKKAVQRLEEQLMKLEVQATDREENKQIALGTSKLNYLDPRITVAWCKKWGVPIEKIYNKTQREKFAWAIDMAD

[0140] EDYEF

[0141] Codon-optimized polynucleotide sequence encoding the truncated form of TOP1 protein from 201-765aa, SEQ ID NO:4:

[0142] CAAAAATGGAAGTGGTGGGAAGAAGAGAGGTACCCGGAGGGCATCAAGTGGAAGTTCCTGGAACATAAGGGTCCGGTGTT

[0143] TGCTCCGCCGTATGAGCCGCTGCCGGAGAACGTCAAATTCTACTATGATGGTAAAGTTATGAAGTTGTCCCCGAAAGCGG

[0144] AAGAAGTGGCGACCTTTTTCGCGAAGATGCTGGACCATGAATACACCACCAAAGAGATCTTTCGCAAGAACTTTTTTAAG

[0145] GACTGGCGAAAGGAGATGACCAACGAGGAGAAGAACATTATAACTAATCTGAGTAAGTGCGATTTCACCCAGATGAGCCA

[0146] GTATTTCAAGGCGCAGACCGAAGCACGCAAACAAATGTCTAAAGAGGAAAAGTTGAAGATCAAAGAGGAGAACGAGAAGT

[0147] TGCTGAAAGAGTACGGCTTTTGTATTATGGATAATCACAAAGAACGTATCGCCAATTTCAAAATCGAGCCGCCTGGCCTG

[0148] TTCCGTGGCCGTGGTAACCACCCGAAAATGGGTATGCTCAAGCGTCGTATCATGCCAGAAGATATTATCATTAACTGCAG

[0149] CAAAGACGCTAAAAGTACCGAGCCCGCCACCCGGCCATAAGTGGAAGGAGGTGCGTCACGATAATAAGGTGACCTGGCTGG

[0150] TTTCCTGGACCGAAAACATTCAAGGTAGCATCAAATACATTATGCTGAACCCGTCGAGCCGTATCAAAGGTGAAAAGGAC

[0151] TGGCAGAAATACGAGACAGCCCGCAGACTGAAGAAATGTGTGGATAAGATCCGTAACCAGTATAGAGAGGGACTGGAAGTC

[0152] CAAGGAAATGAAAGTGCGCCAGCGTGCTGTGGCCCTGTACTTCCATCGACAAATTGGCGCTGCGTGCCCGGTAATGAGAAGG

[0153] AGGAGGGCGAGACTGCTGATACCGTTGGCTGCTGCAGCCTGCGTGTGTGAGCACATTAATCTGCACCCGGAACTGGATGGC

[0154] CAGGAATACGTTGTTGAGTTCGACTTCTTGGGTAAGGACTCAATCCGTTATTACAATAAAGTCCCGGTTGAGAAGCCGGT

[0155] TTTTAAAACCTGCAGTTGTTCATGGAAAACAAACAACCGGAGGATGACTTATTTGATCGTCTGAACACCGGTATTCTGA

[0156] ACAAGCACCTGCAAGATTTAATGGAAGGTCTGACGGCAAAGGTCTTTCGCACCTACAACGCTAGCATTACCCTGCAACAG

[0157] CAGTTGAAAGAACTGACTGCGCCTGAATGAAAATATCCCGGCAAAATTTTTGTCGTATAACCGGCGAATCGTGCGGTCGC

[0158] GATTCTTTGCAATCATCAACGCGCGCCGCCGAAGACCTTTGAAAAATCCATGATGAATTTGCAAACGAAAATCGACGCGA

[0159] AAAAAGAACAGCTGGCTGACGCGCGTCGTGATCTGAAATCTGCAAAAGCGGACGCGAAGGTGATGAAGGACGCCAAGACG

[0160] AAGAAGGTGGTTGAGAGCAAGAAGAAGGCGGTGCAGCGCTTAGAAGAGCAGCTTATGAAATTGGAAGTTCAGGCAACCGA

[0161] TCGCGAAGAGAACAAGCAAATTGCGCTCGGCACCAGCAAGCTGAACTATCTGGACCCGCGTATCACGGTTGCCTGGTGTA

[0162] AAAAATGGGGGGTGCCGATCGAAAAAATCTATAACAAAACGCAACGTGAGAAATTCGCCTGGGCAATTGATATGGCTGAC

[0163] GAAGACTACGAATTT

[0164] Codon-optimized polynucleotide sequence encoding the truncated TOP1 protein 201-755aa, SEQ ID NO:5:

[0165] CAAAAATGGAAGTGGTGGGAAGAGGAAAGGTACCCGGAGGGTATTAAGTGGAAGTTTCTGGAGCACAAGGGTCCGGTGTT

[0166] TGCGCCGCCGTATGAACCGCTGCCGGAGAATGTTAAATTTTATTATGACGGTAAAGTTATGAAATTGAGTCCGAAGGCTG

[0167] AAGAAGTTGCGACGTTTTTTGCGAAGATGCTGGACCATGAATACACCACTAAGGAAATTTTTCGTAAAAATTTCTTCAAA

[0168] GACTGGCGTAAAGAAATGACCAACGAAGAAAAAAATATCATTACAAACTTGTCTAAGTGCGATTTCACCCAGATGTCCCA

[0169] ATATTTCAAAGCACAGACGGAAGCGCGTAAACAGATGAGCAAGGAGGAAAAGCTCAAAATCAAGGAGGAGAACGAAAAAT

[0170] TGCTGAAGGAGTACGGCTTTTGCATCATGGATAACCACAAAGAACGTATAGCGAACTTCAAGATTGAGCCGCCGGGTCTG

[0171] TTTCGTGGTCGTGGCAACCATCCGAAAATGGGTATGCTTAAACGTCGCATCATGCCGGAGGACATCATTATCAACTGCTC

[0172] TAAGGACGCCCAAGGTCCCAAGCCCGCCGCCTGGTCATAAGTGGAAGGAAGTGCGTCATGATAATAAGGTGACCTGGCTGG

[0173] TTAGCTGGACCGAAAACATTCAGGGCTCGATCAAATACATCATGCTGAATCCAAGCAGCCGTTAAAGGCGAAAAGGAC

[0174] TGGCAGAAATACGAGACTGCGAGAAGATTAAAGAAATGCGTTGATAAAATTCGTAACCAGTATCGTGAAGACTGGAAGTC

[0175] CAAAGAGATGAAGGTGCGCCAACGCGCGGTAGCACTGTACTTCATCGACAAACTGGCCTTGCGAGCGGGTAATGAGAAGG

[0176] AGGAGGGCGAAACCGCCGATACCGTGGGTTGTTGTAGCCTGCGTGTCGAGCACATTAACCTGCACCCGGAATTGGACGGC

[0177] CAAGAATACGTGGTGGAGTTTGACTTCCTCGGCAAAGATAGCATCCGTTACTACAACAAAGTTCCGGTCGAGAAACGCGT

[0178] GTTTAAAAACCTCCAATTGTTCATGGAAAACAAACAGCCGGAAGACGACTTGTTCGATCGCCTGAACACCGGTATTCTGA

[0179] ATAAGCACCTGCAGGATCTGATGGAAGGCCTGACCGCGAAGGTTTTCCGCACCTATAACGCGTCTATCACTCTGCAACAA

[0180] CAACTCAAAGAGTTGACGGCTCCGGATGAGAATATCCCGGCAAAGATCTTGAGCTACAACCGTGCTAACCGTGCGGTTGC

[0181] GATCCTTTGCAACCACCAGCGCGCACCACCGAAAACCTTCGAGAAGTCCATGATGAATTTGCAAACCAAAATTGATGCCA

[0182] AGAAGGAGCAGTTGGCAGACGCTCGTCGCGATCTGAAGTCAGCGAAAGCGGATGCCAAAGTCATGAAAGACGCGAAGACC

[0183] AAAAAGGTGGTTGAAAGCAAAAAAAAGGCGGTGCAACGTCTGGAGGAGCAGCTTATGAAACTGGAGGTGCAGGCTACGGA

[0184] CCGCGAGGAGAATAAGCAGATCGCTCTGGGCACCTCCAAGCTGAATTATCTGGATCCGCGTATTACCGTTGCCTGGTGTA

[0185] AAAAGTGGGGTGTTCCGATCGAGAAGATCTATAACAAGACCCAACGTGAAAAATTCGCGTGGGCA

[0186] Codon-optimized polynucleotide sequence encoding truncated TOP1 protein 188-765aa, SEQ ID NO:6

[0187] CCCGATAATAAAAAAAAGAAGCCAAAAAAAGAGGAGGAGCAGAAGTGGAAATGGTGGGAGGAAGAGCGCTACCCGGAGGG

[0188] TATTAAGTGGAAATTCCTGGAACATAAAGGTCCGGTGTTCGCTCCGCCTTATGAACCGCTGCCGGAAAATGTTAAATTTT

[0189] ATTATGATGGTAAAGTGATGAAACTCTCCCCGAAAGCGGAAGAGGTGGCGACCTTTTTTGCGAAAATGCTGGACCACGAA

[0190] TACACCACCAAGGAAATTTTTCGTAAGAATTTTTTCAAAGACTGGCGTAAGGAAATGACGAACGAAGAAAAGAACATCAT

[0191] CACGAACCTGTCGAAGTGCGATTTTACTCAGATGAGCCAGTACTTCAAAGCGCAGACGGAAGCACGTAAACAAATGTCCA

[0192] AGGAGGAGAAGTTGAAAATCAAGGAGGAGAACGAGAAATTACTGAAAGAGTACGGCTTTTGTATTATGGACAACCACAAA

[0193] GAGCGCATTGCGAACTTCAAGATCGAGCCGCCGGGCCTGTTCAGAGGCCGTGGCAATCACCCTAAGATGGGTATGCTGAA

[0194] ACGTCGCATTATGCCGGAAGATATTATTATCAACTGCAGCAAGGACGCGAAGGTGCCGTCTCCGCCACCAGGTCATAAGT

[0195] GGAAAGAAGTGCGCCATGACAACAAGGTCACCTGGCTGGTTAGCTGGACCGAAAACATCCAAGGTAGCATCAAATACATC

[0196] ATGCTGAACCCGAGCTCCCGCATCAAGGGCGAGAAGGATTGGCAAAAATATGAAACAGCGCGTCGTTTGAAGAAGTGCGT

[0197] GGATAAGATTCGTAATCAGTATCGAGAGGATTGGAAGTCTAAGGAGATGAAGGTGCGCCAGCGTGCGGTTGCCCTCTACT

[0198] TCATCGACAAACTGGCGCTGCGTGCGGGTAATGAAAAAGAGGAGGGCGAAACGGCCGACACCGTAGGTTGTTGTTCACTG

[0199] CGTGTTGAGCACATTAATCTGCACCCGGAGCTAGACGGCCAAGAATACGTTGTCGAGTTCGACTTCCTGGGTAAAGACAG

[0200] CATTCGTTACTACAATAAGGTGCCGGTTGAAAAACGCGTGTTCAAAAATTTGCAACTGTTTATGGAAAACAAGCAACCGG

[0201] AAGACGATTTATTCGACCGTCTGAACACCGGCATTCTGAACAAACACCTTCAAGATCTGATGGAAGGCCTGACCGCGAAA

[0202] GTCTTTCGGACCTACAACGCGTCCATCACTTTGCAACAACAACTGAAGGAGCTGACGGCTCCGGATGAAAATATCCCGGC

[0203] AAAGATCCTGAGCTATAACCGCGCTAATCGTGCGGTGGCCATCCTTTGCAACCATCAGCGTGCTCCGCCGAAAACCTTTG

[0204] AAAAGTCTATGATGAACCTGCAGACCAAAATTGATGCTAAAAAAGAGCAGTTAGCCGATGCTCGTCGTGATCTGAAAAGC

[0205] GCGAAGGCTGACGCCAAGGTCATGAAGGACGCAAAAACCAAAAAGGTGGTTGAAAGCAAGAAGAAGGCCGTTCAGCGCTT

[0206] GGAGGAACAGCTGATGAAACTAGAGGTTCAGGCAACCGATCGTGAAGAGAATAAACAGATTGCACTGGGAACCAGCAAGT

[0207] TGAACTATTTGGATCCGCGTATCACCGTTGCGTGGTGCAAAAAATGGGGTGTTCCGATCGAGAAAATTTATAACAAGACC

[0208] CAGAGAGAAAAGTTTGCATGGGCAATCGATATGGCGGACGAGGACTACGAGTTC

[0209] Example 2: Expression of target gene

[0210] The monoclonal clone prepared in Example 1 was picked and aseptically inoculated into TB medium containing 100 μg / mL kanamycin resistance. The culture was shaken at 37°C and 220 rpm until the OD600 was between 0.6 and 0.8. IPTG was used for induction and the culture was shaken at 18°C overnight. Equal amounts of the bacterial solution were ultrasonically disrupted and centrifuged. The supernatant and precipitate were collected and then identified by SDS-PAGE. The identification results are shown in Figure 2. Figure 1 .

[0211] like Figure 1 As shown, columns 1-2: supernatant and precipitate of E. coli expressing 201-755aa truncation; columns 3-4: supernatant and precipitate of E. coli expressing 201-765aa truncation; columns 5-6: supernatant and precipitate of E. coli expressing 188-765aa truncation.

[0212] Combined with the predicted molecular weight of 69.09KD, the results showed that the expression effect of the 201-765aa truncation in column 3-4 was significantly better than that of the 188-765aa truncation and the 201-755aa truncation.

[0213] Example 3: Purification of the expression product

[0214] 1.5L bacterial liquid (201-765aa truncated form) was cultured in a shake flask and the wet weight of the collected bacteria was 69.09g. About 4g of bacteria were weighed and resuspended on ice in 20ml Lysis Buffer. After ultrasonic disruption, centrifugation was performed at 20000rpm and 4℃ for 30min. The supernatant was collected and filtered through a 0.22μm needle filter to obtain the filtered bacterial liquid. After filtration, the protein eluted with 50mM Tris-HCl, 50mM NaCl, and 200mM imidazole at pH 7.0 was the target protein. The electrophoresis pattern is shown in the figure below. Figure 2 shown.

[0215] The target protein expression content was calculated to be 66.0 mg / L, and the purity reached 95%.

[0216] Example 4: Chemiluminescence identification of target protein activity

[0217] (1) Preparation of antigen-antibody sandwich complex: The Sc1-70 antigen expressed by the present invention and the magnetic strain coated with the Sc1-70 antigen (purchased from other companies) are used as coating materials, and the Sc1-70 monoclonal antibody and the secondary antibody (Phipeng) are added and reacted under incubation conditions. The antibody captures the antigen in the sample to form a complex.

[0218] (2) Detection and Reading: After the incubation period, a magnetic field is applied to precipitate the complex. The supernatant is removed and the precipitated complex is washed with a cleaning solution. The waste liquid is aspirated to remove any material not bound to the magnetic particles. The reaction cup is then placed into the measurement chamber. The instrument automatically pumps in two excitation solutions, causing the complex to produce a chemiluminescent signal, and the luminescence intensity is measured. Following the above detection method, the antigen performance of two different sources of Sc1-70 antigen was tested.

[0219] The results of the chemiluminescence method are shown in Tables 1 and 2. The luminescence value of the commercially available Sc1-70 antigen can reach up to 4.56 million, while the luminescence value of the Sc1-70 antigen of the present invention can reach up to 4.25 million, indicating that both antigens have good performance. The linear regression equation of concentration and RLU value is as follows: y1 = 1E + 09x + 370549, R 2 =0.9162;y2=1E+09x+256579,R 2 =0.9592, indicating that the linearity of the antigen of the present invention is better than that of the commercially available antigen.

[0220] Table 1

[0221]

[0222] Table 2

[0223]

[0224] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A truncated form of the SC1-70 antigen, characterized in that: The SC1-70 antigen truncation is shown in SEQ ID NO.

3.

2. An isolated polynucleotide encoding the SC1-70 antigen truncation according to claim 1, characterized in that The polynucleotide is codon-optimized, and the polynucleotide sequence is shown in SEQ ID NO.

4.

3. An expression vector, characterized in that The expression vector comprises the polynucleotide according to claim 2, and the expression vector is an Escherichia coli expression vector.

4. The expression vector according to claim 3, characterized in that The expression vector includes a polynucleotide sequence for expressing a His×6 tag.

5. The expression vector according to claim 3, characterized in that The expression vector is pET-28a(+).

6. A host cell, characterized in that The host cell is Escherichia coli; wherein, The host cell comprises the expression vector according to any one of claims 3 to 5; or The polynucleotide according to claim 2 is integrated into the genome of the host cell.

7. A method for preparing a truncated form of the SC1-70 antigen, characterized in that: The method comprises the steps of: Transforming an Escherichia coli host cell with the polynucleotide vector according to claim 2; The host cells are cultured to express the SC1-70 antigen truncation.

8. The method according to claim 7, characterized in that The host cells were cultured using SB, TB or SOC medium.

9. The method according to claim 7, characterized in that When the host cells are cultured, they are induced by IPTG to express the target protein.

10. A kit, characterized in that The kit comprises: the polynucleotide according to claim 2; or The expression vector according to any one of claims 3 to 5; or The host cell according to claim 6; or An SC1-70 antigen truncate prepared by the method according to any one of claims 7 to 9.

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