CN115960203A - CEA truncation, preparation method and application thereof - Google Patents

Abstract

The application discloses a CEA truncated body and a preparation method and application thereof. A CEA preparation method based on genetic engineering technology is developed in the application, the method obtains a CEA truncated body by carrying out truncation modification on CEA protein, and a polynucleotide encoding the CEA truncated body optimized by preference of human synonymous codons is introduced into a vector to construct a CEA eukaryotic expression system, so that the expression quantity and activity of soluble target protein are improved, and the separation and purification are easy.

Description

CEA truncated body, its preparation method and application

technical field

The invention relates to the field of genetic engineering, in particular to a CEA truncated body, its preparation method and application.

Background technique

Carcinoembryonic antigen (CEA) is a cell surface adhesion molecule of the immunoglobulin superfamily. CEA consists of 702 amino acids and is mainly anchored to the cell membrane by phosphatidylinositol (GPI). The determination of serum CEA has great significance for the prognosis and treatment of patients with malignant diseases, especially for patients with colorectal cancer. Follow-up assays can monitor cancer progression, regression, or recurrence in treated patients. Clinically, the dynamic detection of CEA has been used as one of the important monitoring indicators for the early diagnosis and prognosis evaluation of positive tumors. In recent years, studies have found that CEA can also be recognized by specific T cells in the human body and induce anti-tumor immune responses, so it has been used in the immunobiological therapy of CEA-positive tumors.

Some preparation methods of CEA have been disclosed in the prior art, such as natural extraction method and prokaryotic system expression method. Natural extraction requires the use of hypochlorous acid to precipitate tumor cell extracts from colon cancer liver metastases, and then obtains them by ion exchange chromatography and affinity chromatography, but the CEA obtained by this method may be a mixture of several molecules with different properties, Separation is difficult. The expression of prokaryotic expression system lacks post-translational processing and modification of human CEA, and has the disadvantages of low biological activity and low sensitivity of the product.

In order to overcome the defects of prokaryotic system expression, some researchers have developed a CEA preparation method based on eukaryotic expression system, but its expression level is extremely low, making it difficult to industrialize.

Therefore, there is still a need in the art to find a method that can express a large amount of human CEA protein with high biological activity.

Contents of the invention

The purpose of the present invention is to provide a CEA truncation body.

Another object of the present invention is to provide a method for preparing recombinant CEA truncations.

Another object of the present invention is to provide a polynucleotide sequence encoding a CEA truncation.

Another object of the present invention is to provide a vector adapted to the polynucleotide sequence encoding the CEA truncation.

Another object of the present invention is to provide a vector containing a polynucleotide sequence encoding an A-CEA truncate.

Another object of the present invention is to provide a kit comprising a polynucleotide sequence encoding a CEA truncation.

In order to solve the above technical problems, the first aspect of the present invention provides a CEA truncation body, and the CEA truncation body is selected from any of the following:

(i) a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.3;

(ii) a polypeptide having an amino acid sequence with greater than 95% homology with the sequence shown in SEQ ID NO.1 or SEQ ID NO.3.

In a second aspect, the present invention provides a polynucleotide encoding a CEA truncation body, the polynucleotide is codon-optimized, and the polynucleotide is selected from any of the following:

(i) have the polynucleotide of sequence as shown in SEQ ID NO.2 or SEQ ID NO.4;

(ii) have a polynucleotide with greater than 95% homology with the sequence shown in SEQ ID NO.2 or SEQ ID NO.4; and

(iii) has a polynucleotide complementary to the polynucleotide sequence described in (i) or (ii).

In the third aspect of the present invention, an expression vector is provided, and the expression vector includes the polynucleotide provided in the second aspect of the present invention.

In some preferred schemes, the expression vector is a mammalian expression vector, more preferably pcDNA3.4.

In some preferred schemes, the expression vector includes a polynucleotide sequence encoding protein A, more preferably, in the expression vector, the 5' end of the polynucleotide is linked with a polynucleotide encoding protein A sequence.

In some preferred schemes, the polynucleotide sequence encoding protein A is shown in SEQ ID NO.6.

In some preferred schemes, the expression vector includes a polynucleotide sequence encoding an A-CEA truncated fusion protein.

In some preferred schemes, the polynucleotide sequence encoding the A-CEA truncated fusion protein is selected from any of the following:

(i) have the polynucleotide of sequence as shown in SEQ ID NO.11 or SEQ ID NO.9;

(ii) have a polynucleotide with greater than 95% homology with the sequence shown in SEQ ID NO.11 or SEQ ID NO.9; and

(iii) has a polynucleotide complementary to the polynucleotide sequence described in (i) or (ii). In some preferred schemes, the expression vector includes a polynucleotide sequence expressing a His×6 tag. More preferably, in the expression vector, the 3′ end of the polynucleotide is linked with a His×6 tag polynucleotide sequence.

In some preferred schemes, the expression vector includes a kozak sequence before the start codon of the polynucleotide, more preferably, the kozak sequence is shown in SEQ ID NO.7.

In the fourth aspect of the present invention, a host cell is provided, and the host cell includes the expression vector provided in the third aspect of the present invention; or

The polynucleotide provided in the second aspect of the present invention is integrated in the genome of the host cell.

In some preferred schemes, the host cells are CHO cells (Chinese Hamster Ovary) or 293F cells, most preferably 293F cells.

The fifth aspect of the present invention provides a method for preparing a CEA truncation, the method comprising the steps of: cultivating the host cell described in the fourth aspect of the present invention to express the target protein; and

Isolating the target protein to obtain the CEA truncated body;

Wherein, the target protein has an amino acid sequence such as the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

In some preferred schemes, the host cells are obtained by transforming CHO cells or 293F cells with a plasmid containing the polynucleotide according to the second aspect of the present invention.

In some preferred schemes, the conversion time is 15 to 24 hours.

In some preferred schemes, the host cells are cultured in a culture system containing enhancers and adjuvants.

In some preferred embodiments, the host cells are cultured in an environment with a temperature of 35 to 37°C.

In some preferred schemes, the host cells are cultured in an environment with a _CO2 concentration of 8%.

In some preferred schemes, the host cells are cultured until the survival rate is not lower than 50%, preferably 60%, and the supernatant is collected by centrifugation to obtain the supernatant containing the target protein.

In some preferred schemes, the step of isolating the protein of interest comprises:

The supernatant containing the target protein is passed through the chromatographic column for elution, and the eluate is collected.

In some preferred schemes, the chromatography column is a Ni-column affinity chromatography column.

The sixth aspect of the present invention provides a kit, said kit comprising: the CEA truncate as provided in the first aspect of the present invention; or

The polynucleotide provided by the second aspect of the present invention; or

The expression vector as provided in the third aspect of the present invention; or

The host cell as described in the fourth aspect of the present invention.

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

(1) The present invention has developed a CEA preparation method based on genetic engineering technology. In this method, the CEA truncation body is obtained by truncation and transformation of the CEA protein. The polynucleotide is introduced into the vector, and the CEA eukaryotic expression system is constructed, which improves the expression and activity of the soluble target protein, and is easy to separate and purify;

(2) In a preferred embodiment of the present invention, the gene translation efficiency is enhanced by adding the kozak sequence before the initiation codon ATG;

(3) In a preferred embodiment of the present invention, the secretion efficiency and expression amount of the CEA protein are further improved by linking the polynucleotide sequence encoding the A-CEA truncated fusion protein to the vector.

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 specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

One or more embodiments are exemplified by pictures in the accompanying drawings, and these exemplifications are not intended to limit the embodiments.

Fig. 1 is an electrophoresis diagram of CEA prepared in an embodiment of the present invention.

Detailed ways

After extensive and in-depth research, the inventors have established a method for high-efficiency expression and preparation of exocrine human CEA. By designing a CEA truncated body and further optimizing the synonymous codon preference of the gene sequence encoding it, the obtained A polynucleotide sequence that expresses a CEA truncation can be secreted.

Preferably, the gene sequence encoding the CEA truncation body and the gene sequence encoding the A protein are spliced to obtain a polynucleotide sequence encoding the A-CEA truncation body fusion protein, which further increases the expression level of the transient expression vector.

Preferably, by adopting a pcDNA3.4 vector suitable for secreted protein expression, and adding a kozac sequence before the start codon ATG, the expression level of the protein is further increased.

Preferably, the CEA protein with high biological activity is successfully obtained through the transient expression of the 293F system.

Obtain the target gene/acquire the nucleic acid sequence related to the target protein

The full-length nucleotide sequence or its fragments of the target protein or its elements in the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as Template, amplified to obtain related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Primers for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

In one embodiment of the present invention, the target gene sequence information can be obtained by analyzing the amino acid sequence of the CEA protein through the NCBI database.

In one embodiment of the present invention, truncation is carried out on the basis of the amino acid sequence of the CEA protein, and 34 amino acids at the N-terminus are truncated to obtain the amino acid sequence shown in SEQ ID NO: 1, which is named truncation body A. It conducts gene sequence analysis, and the gene sequence encoding it can be obtained.

In one embodiment of the present invention, truncation is performed on the basis of the amino acid sequence of the CEA protein to obtain the amino acid sequence shown in SEQ ID NO: 3, which is named truncated body B, and its gene sequence analysis can obtain the encoded its gene sequence.

Synonymous codon preference optimization

To overcome the potential problem of reduced yield when expressing heterologous proteins in mammalian cells, the present invention relates to polynucleotide sequences optimized for synonymous codon bias. Truncating the CEA protein, preferably truncating 34 amino acids at the N-terminus, and then performing sequence analysis on it to obtain the polynucleotide sequence encoding it, and then optimizing the synonymous codon preference. The polynucleotide sequence (SEQ ID NO:2) can express the same amino acid sequence as the target protein truncated A, but the stability and efficiency of the expression process are improved, and the final target protein maintains a high activity.

The present invention also relates to a polynucleotide with a homology greater than 95% of the sequence shown in SEQ ID NO:2; and a polynucleotide complementary to the sequence shown in SEQ ID NO:2.

In a preferred embodiment of the present invention, the target protein is truncated, preferably after truncating 34 amino acids at the N-terminus and GPI at the C-terminus at the same time to obtain a truncated body B, and then sequence analysis is performed on the truncated body to obtain the truncated body encoding B. The polynucleotide sequence is then optimized for synonymous codon preference, and the polynucleotide sequence (SEQ ID NO: 4) optimized for synonymous codon preference can express the same amino acid sequence as the target protein truncated body B. SEQ ID NO:4 increases the expression of the target protein by further intercepting the C-terminal GPI.

Carrier of target gene

The invention also relates to vectors comprising the polynucleotides of the invention. "Vector" in the present invention means 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. A vector segment may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector can be any expression vector that is synthetic or convenient for recombinant DNA manipulation, and the choice of the vector usually depends on the host cell into which the vector is to be introduced. Thus, a vector may be an autonomously replicating vector, ie a vector, which exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome of the host cell and replicates with the chromosome into which it has integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the present invention, pcDNA3.4 is selected as the carrier, which is more suitable for secreting proteins and promoting the improvement of expression efficiency.

In a preferred embodiment of the present invention, the expression vector includes a polynucleotide sequence (SEQ ID NO: 6) encoding protein A.

In a preferred embodiment of the present invention, the expression vector comprises a polynucleotide series (SEQ ID NO: 9 or SEQ ID NO: 11) encoding the A-CEA truncation body. The secretion efficiency and expression amount of the CEA truncation can be improved by adding the polynucleotide series encoding the A-CEA truncation to the expression vector.

In a preferred embodiment of the present invention, the expression vector includes a polynucleotide sequence expressing a His×6 tag. More preferably, in the expression vector, the 3′ end of the polynucleotide is linked with a His× 6 tagged polynucleotide sequences. The expression vector connected with the polynucleotide encoding the His×6 tag is helpful for the separation and purification of the target protein.

In a preferred embodiment of the present invention, the expression vector includes a kozak sequence before the start codon of the polynucleotide, more preferably, the kozak sequence is shown in SEQ ID NO.7. The expression vector connected with the kozak sequence can optimize the environment before the start codon, making it more suitable for the translation of the target gene and enhancing the translation efficiency of the target gene.

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 technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Exemplarily, the DNA endonuclease is used to cut the carrier DNA molecule into a linear molecule that can be connected with the foreign gene, and then the codon-optimized target gene fragment is connected to the carrier, and the sticky end connection with a single enzyme cutting site can be selected , Directional cloning of double-digested fragments, sticky-end ligation of different restriction enzyme sites, blunt-end ligation, artificial adapter ligation or ligation with oligonucleotide ends to achieve the insertion of exogenous DNA fragments.

Transform host cells with the vector containing the gene of interest

The present invention also relates to host cells produced by genetic engineering using the vector or fusion protein coding sequence of the present invention. The vector containing the codon-optimized gene of interest can be inserted, transfected or otherwise transformed into host cells by known methods, so as to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest . A "host cell" in the present invention is a cell into which exogenous polynucleotides and/or vectors have been introduced. The host cell can be a eukaryotic host cell or a prokaryotic host cell, and the host cell is preferably a mammalian cell, and is preferably a CHO cell or a 293F cell, more preferably a 293F cell.

Method for preparing target protein

The present invention also relates to a method for preparing the target protein, and the polynucleotide sequence of the present invention can be used to express or produce the recombinant protein. Generally speaking, there are the following steps:

(1) Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Separation and purification of protein from culture medium or cells.

Wherein, step (1) transforming or transducing suitable host cells 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 CHO cells or 293F cells, alternatively, use Calcium phosphate co-precipitation method, electroporation method, microinjection method or liposome-mediated gene transfer method, etc. For example, the specific operation of electroporation is to mix exogenous DNA and host cells in an electroporation cup. Under the action of high-frequency current, many small holes appear in the cell membrane to allow exogenous DNA to enter the host cells.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. In one embodiment of the present invention, the host cells are cultured in an environment of 8% CO ₂ and 36.5°C. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time. In order to promote the expression of the target protein, in a preferred embodiment of the present invention, the host cells are cultivated using a culture system containing enhancers and auxiliary materials.

The protein in the above method may be expressed inside the cell, or on the cell membrane, or secreted outside the cell. Proteins can be isolated and purified by various separation methods by taking advantage of their physical, chemical and other properties, if desired. 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 precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. In one embodiment of the invention, the protein of interest is isolated using affinity chromatography.

In this disclosure, use of any exemplary or exemplary language (eg, "") provided with respect to certain embodiments herein is for the purpose of better presenting the invention only and does not limit the scope of the invention otherwise claimed. scope. Nothing in the text should be construed as indicating any non-recited element in the claims indispensable to the practice of the invention.

If the definition or use of a term in a cited document is inconsistent or inconsistent with the definition of the term described herein, the definition of the term described herein applies instead of the definition of the term in the cited document.

Various terms used in this article are as follows. To the extent 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.

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

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 can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.

The present invention also relates to variants of the above polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide.

As used herein, the term "codon optimization" refers to avoiding codon usage differences based on the actual expression or production of proteins (including Escherichia coli, yeast, mammalian blood cells, plant cells, insect cells, etc.) A way to improve the efficiency of gene synthesis by using low-utilization or rare codons.

As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (ie identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. Align the nucleotide or amino acid sequences of polynucleotides or polypeptides, score the number of positions in the aligned polynucleotides or polypeptides that contain the same nucleotide or amino acid residue, and compare this with the aligned polynucleotides or polypeptides Compare the number of positions containing different nucleotide or amino acid residues in . Polynucleotides may differ at a position, for example, by the inclusion of different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertion or deletion of one or two nucleotides in the polynucleotide ). Polypeptides may differ at one position, for example, by the inclusion of amino acids (ie, substitutions or variations) or deletions of amino acids (ie, insertions or deletions of amino acids in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

As used herein, the terms "sequence complementarity" and "reverse sequence complementarity" are used interchangeably to refer to a sequence that is oriented in the opposite direction to an original polynucleotide sequence and that is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAT.

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. Following expression, it can be harvested, ie, the host cells or the expression product are recovered.

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the present invention will be further described below in conjunction with specific embodiments. 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. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.

Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the application belongs. Exemplary implementation of the application.

Embodiment 1, construct CEA plasmid and transfect host cell

(1) Obtain the amino acid sequence of human CEA, remove the signal peptide of 34 amino acids at the N-terminal to obtain CEA truncated body A, its amino acid sequence is shown in SEQ ID NO: 1, analyze it to obtain the gene sequence, and perform human The source synonymous codon bias was optimized to obtain the gene sequence SEQ ID NO: 2 optimized by the synonymous codon bias.

Further truncation was carried out on the basis of SEQ ID NO: 1 to obtain CEA truncated body B. After gene sequence analysis and synonymous codon preference optimization, the gene sequence SEQ ID NO: 4 was obtained.

The N-terminal of SEQ ID NO: 2 and SEQ ID NO: 4 is connected to the polynucleotide sequence encoding A protein SEQ ID NO: 6, the carrier is pcDNA3.4, and the C-terminal is connected to the (His) 6 tag, respectively and commissioned by Nanjing Kings Ruisynthetic recombinant expression plasmid A (contains the sequence encoding A-CEA truncation A fusion protein) and plasmid B (contains the sequence encoding A-CEA truncation B fusion protein).

Truncated body A amino acid sequence SEQ ID NO: 1

KLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQ

NDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLS

NGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVN

GTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTY

LWWVNNQSLPVSPRLQLSNDNRTLTLSLVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLS

LSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVE

DKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPD

TPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITV

SASGTSPGLSAGATVGIMIGVLVGVALI

Optimized polynucleotide sequence encoding truncated body A SEQ ID NO: 2

AAGCTGACCATCGAGAGCACACCTTTCAACGTCGCTGAAGGCAAGGAAGTACTGCTGCTTGTGCACAACTTGCCACAACA

CCTGTTCGGCTACTCCTGGTACAAGGGCGAGCGGGTGGACGGTAACCGGCAGATCATCGGCTACGTGATCGGAACCCAGC

AAGCCACGCCTGGCCCCGCCTACAGCGGTCGGGAAATCATCTACCCCAATGCCAGCCTGCTGATCCAAAATATCATCCAG

AATGATACAGGCTTTCTACACCCTGCACGTGATTAAGTCCGACCTGGTGAATGAGGAAGCCACCGGCCAGTTCAGAGTGTA

TCCTGAACTGCCTAAACCCAGCATCAGCAGCAACAACTCTAAGCCAGTTGAGGACAAGGACGCCGTGGCCTTCACCTGTG

AACCCGAGACACAGGACGCCACATACCTGTGGTGGGTCAACAACCAGAGCCTGCCAGTGAGCCCTCGGCTGCAGCTGAGT

AACGGCAATAGAACACTGACCCTGTTTAACGTGACACGGAACGACACCGCCAGCTACAAGTGCGAAACCCAAAACCCTGT

GAGCGCCAGACGGAGCGACAGCGTGATCCTGAACGTGCTGTACGGCCCTGACGCTCCTACAATCAGCCCTCTGAATACCA

GCTATAGAAGCGGGGAAAACCTGAACCTGTCTTGCCACGCCGCCAGCAACCCTCCTGCTCAGTACAGCTGGTTCGTGAAC

GGCACCTTTCAGCAGAGCACCCAGGAGCTCTTCATCCCCAACATCACAGTGAACAACAGCGGATCTTATACCTGTCAGGC

CCACAACTCCGATACCGGCCTGAACAGGACCACCGTGACTACCATCACCGTCTACGCCGAGCCTCCTAAGCCCTTCATCA

CCAGCAACAACAGCAATCCTGTTGAGGACGAGGATGCCGTGGCCCTGACCTGTGAGCCAGAGATCCAGAACACCACATAC

CTGTGGTGGGTGAACAACCAGAGCCTCCCCGTGTCCCCTAGACTGCAGCTGAGCAACGACAACAGAACACTGACACTGCT

GAGCGTGACAAGAAACGACGTGGGACCCTACGAGTGCGGCATCCAGAACGAGCTGAGCGTGGACCACTCAGACCCTGTGA

TCCTGAACGTACTGTATGGCCCCGATGACCCTACCATCTCACCCCTCTACACCTACTACAGACCTGGCGTGAACCTGTCT

CTGAGCTGTCATGCTGCTTCTAATCCTCCTGCTCAGTACAGCTGGCTGATCGACGGCAACATCCAGCAGCACACCCAGGA

GCTGTTCATTAGCAATATCACGGAAAAAAACAGCGGCCTGTACACCTGCCAGGCCAACAACAGCGCCTCCGGCCACTCTA

GAACCACAGTGAAGACCATTACAGTGAGCGCCGAGCTGCCCAAGCCATCCATCAGCAGCAACAACAGCAAACCTGTGGAA

GATAAAGACGCAGTTGCTTTCACATGCGAGCCTGAGGCTCAGAATACCACCTACCTGTGGTGGGTCAATGGCCAGTCTCT

GCCTGTGAGCCCCCGCCTCCAGCTGAGCAATGGAAATAGAACACTGACCCTGTTCAACGTGACCAGAAACGACGCCAGAG

CCTACGTGTGCGGCATCCAGAACTCTGTGTCGGCCAACAGATCAGACCCTGTGACCCTGGATGTGCTGTACGGCCCTGAT

ACCCCCATCATCTCTCCACCTGATTCTTCTTACCTCAGCGGAGCAAACCTGAACCTGAGCTGCCACAGCGCCAGCAACCC

CAGCCCACAGTACTCTTGGCGGATCAACGGAATCCCTCAACAGCACACCCAAGTGCTGTTTATCGCCAAGATCACACCTA

ACAATAACGGCACCTACGCCTGCTTCGTGTCCAACCTGGCTACCGGCAGAAACAACTCGATTGTTAAGAGCATCACCGTG

TCTGCCAGCGGCACATCCCCTGGTCTGAGCGCCGGCGCCACAGTCGGCATCATGATCGGCGTGCTGGTGGGCGTGGCCCT

GATC

Truncated body B amino acid sequence SEQ ID NO: 3

KLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQ

NDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLS

NGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVN

GTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTY

LWWVNNQSLPVSPRLQLSNDNRTLTLSLVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLS

LSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVE

DKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPD

TPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITV

SASGTSPGLSA

Optimized polynucleotide sequence encoding truncated body B SEQ ID NO:4

AAGCTGACCATCGAGTCCACCCCTTTCAATGTGGCTGAGGGCAAGGAGGTGCTGCTGCTGGTGCACAACCTGCCTCAGCA

CCTGTTCGGCTACAGCTGGTACAAGGGCGAGAGAGTGGATGGCAATCGGCAGATCATCGGCTACGTGATCGGCACACAAC

AAGCCACCCCCGGCCCAGCCTACAGCGGACGCGAAATCATCTACCCCAATGCCTCTCTGCTGATCCAGAACATCATTCAA

AACGACACAGGCTTTCTACACCCTGCACGTGATCAAGAGCGACCTGGTGAACGAGGAAGCCACCGGCCAGTTCAGAGTGTA

CCCTGAACTGCCTAAGCCCAGCATCAGCAGCAACAACTCCAAGCCCGTGGAAGACAAAGACGCCGTGGCCTTCACCTGCG

AGCCTGAGACACAGGACGCTACATACCTGTGGTGGGTCAACAACCAGAGCCTGCCAGTGAGCCCTAGACTGCAGCTGAGC

AACGGCAACAGAACCCTGACACTGTTTAACGTGACAAGAAATGACACCGCCAGCTACAAGTGCGAGACACAGAACCCTGT

GAGCGCCAGAAGAAGCGACAGCGTGATCCTGAACGTGCTGTACGGCCCTGACGCCCCTACCATTTCTCCTCTGAACACCT

CTTACCGGAGCGGCGAAAACCTGAACCTGAGCTGCCATGCCGCTTCTAATCCTCCTGCTCAGTACAGCTGGTTCGTGAAT

GGCACCTTCCAGCAGTCTACCCAGGAGCTGTTTATCCCCAACATCACTGTGAACAACAGCGGCAGCTATACATGTCAGGC

CCACAACAGCGACACCGGACTGAACAGGACCACCGTAACCACAATCACCGTGTACGCCGAGCCTCCTAAGCCCTTCATCA

CATCTAACAACTCTAACCCTGTGGAAGATGAGGATGCTGTTGCATTGACCTGCGAGCCTGAGATCCAGAACACCACCTAC

CTGTGGTGGGTGAACAACCAGAGCCTGCCCGTGAGCCCGAGACTGCAGCTGTCTAACGACAACAGGACCCTGACCCTGCT

GAGCGTGACCAGAAATGATGTGGGCCCCTACGAGTGCGGGATCCAGAATGAGCTGAGCGTGGATCACAGCGATCCCGTGA

TTCTGAACGTGCTGTACGGCCCTGATGATCCAACCATCAGCCCTAGCTACACCTACTACAGACCAGGCGTGAACCTGAGC

CTGTCTTGTCACGCCGCCAGCAATCCTCCAGCCCAGTACAGCTGGCTGATCGACGGCAACATCCAGCAACATACCCAGGA

GCTGTTCATCAGCAACATCACGGAAAAGAACTCCGGACTTTATACCTGCCAGGCCAACAATTCTGCCAGCGGCCACTCAA

GAACAACCGTCAAAAACCATCACCGTGAGCGCTGAACTGCCTAAACCCAGCATTTCTAGCAACAACTCTAAACCTGTCGAA

GACAAGGACGCCGTGGCTTTTACATGTGAACCTGAGGCCCAGAACACCACTTATCTGTGGTGGGTCAACGGCCAGAGCCT

GCCCGTGTCCCCCCCGGCTGCAACTGTCTAATGGCAACAGAACCCTGACACTGTTCAACGTGACACGGAACGACGCCAGAG

CCTACGTTTGTGGCATCCAGAATAGCGTGAGCGCCAACCGGAGCGACCCCGTCACCCTGGACGTGCTCTATGGCCCTGAT

ACACCTATCATCAGCCCTCCCGACAGCAGCTACCTGAGCGGCGCCAATCTGAACCTGTCCTGCCACTCTGCTAGCAATCC

TAGCCCACAGTACAGCTGGCGGATCAACGGAATCCCTCAGCAGCACACCCAGGTGCTCTTCATCGCCAAGATCACCCCTA

ACAACAACGGCACCTACGCTTGCTTCGTGTCCAACCTGGCCACAGGACGGAACAACAGCATCGTGAAGTCCATCACAGTG

TCCGCCTCTGGAACCAGCCCTGGCCTGAGTGCC

Protein A amino acid sequence SEQ ID NO:5

MKWVTFISLLFSSAYS

Optimized polynucleotide sequence encoding protein A SEQ ID NO:6

ATGAAGTGGGTGACCTTTCATCAGCCTGCTGTTCAGCTCCGCCTACTCT

kozak sequence SEQ ID NO:7

GCCACC

A-CEA truncated body A fusion protein amino acid sequence SEQ ID NO: 8:

MKWVTFISLLFSSAYSKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGR

EIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLW

WVNNQSLPVSPRLQLSNGNRTLLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLS

CHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDE

DAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDP

TISPSYTYYRPGVNLSLSCHASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSA

ELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLLFNVTRNDARAYVCGIQNSVS

ANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVS

NLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALIHHHHHH

The polynucleotide sequence SEQ ID NO: 9 encoding the A-CEA truncated body A fusion protein:

ATGAAGTGGGTGACCTTTCATCAGCCTGCTGTTCAGCTCCGCCTACTCTAAGCTGACCATCGAGAGCACACCTTTCAACGT

CGCTGAAGGCAAGGAAGTACTGCTGCTTGTGCACAACTTGCCACAACACCTGTTCGGCTACTCCTGGTACAAGGGCGAGC

GGGTGGACGGTAACCGGCAGATCATCGGCTACGTGATCGGAACCCAGCAAGCCACGCCTGGCCCCGCCTACAGCGGTCGG

GAAATCATCTACCCCCAATGCCAGCCTGCTGATCCAAAATATCATCCAGAATGATACAGGCTTCTACACCCTGCACGTGAT

TAAGTCCGACCTGGTGAATGAGGAAGCCACCGGCCAGTTCAGAGTGTATCCTGAACTGCCTAAACCCAGCATCAGCAGCA

ACAACTCTAAGCCAGTTGAGGACAAGGACGCCGTGGCCTTCACCTGTGAACCCGAGACACAGGACGCCACATACCTGTGG

TGGGTCAACAAACCAGAGCCTGCCAGTGAGCCCTCGGCTGCAGCTGAGTAACGGCAATAGAACACTGACCCTGTTTAACGT

GACACGGAACGACACCGCCAGCTACAAGTGCGAAACCCAAAACCCTGTGAGCGCCAGACGGAGCGACAGCGTGATCCTGA

ACGTGCTGTACGGCCCTGACGCTCCTACAATCAGCCCTCTGAATACCAGCTATAGAAGCGGGGAAAACCTGAACCTGTCT

TGCCACGCCGCCAGCAACCCTCCTGCTCAGTACAGCTGGTTCGTGAACGGCACCTTTCAGCAGAGCACCCAGGAGCTCTT

CATCCCCAACATCACAGTGAACAACAGCGGATCTTATACCTGTCAGGCCCACAACTCCGATACCGGCCTGAACAGGACCA

CCGTGACTACCATCACCGTCTACGCCGAGCCTCCTAAGCCCTTCATCACCAGCAACAACAGCAATCCTGTTGAGGACGAG

GATGCCGTGGCCCTGACCTGTGAGCCAGAGATCCAGAACACCACATACCTGTGGTGGGTGAACAACCAGAGCCTCCCCGT

GTCCCCTAGACTGCAGCTGAGCAACGACAACAGAACACTGACACTGCTGAGCGTGACAAGAAACGACGTGGGACCCTACG

AGTGCGGCATCCAGAACGAGCTGAGCGTGGACCACTCAGACCCTGTGATCCTGAACGTACTGTATGGCCCCGATGACCCT

ACCATCTCACCCCTCTTACACCTACTACAGACCTGGCGTGAACCTGTCTCTGAGCTGTCATGCTGCTTCTAATCCTCCTGC

TCAGTACAGCTGGCTGATCGACGGCAACATCCAGCAGCACACCCAGGAGCTGTTCATTAGCAATATCACGGAAAAAAACA

GCGGCCTGTACACCTGCCAGGCCAACAACAGCGCCTCCGGCCACTCTAGAACCACAGTGAAGACCATTACAGTGAGCGCC

GAGCTGCCCAAGCCATCCATCAGCAGCAACAACAGCAAACCTGTGGAAGATAAAGACGCAGTTGCTTTCACATGCGAGCC

TGAGGCTCAGAATACCACCTACCTGTGGTGGGTCAATGGCCAGTCTCTGCCTGTGAGCCCCCGCCTCCAGCTGAGCAATG

GAAATAGAACACTGACCCTGTTCAACGTGACCAGAAACGACGCCAGAGCCTACGTGTGCGGCATCCAGAACTCTGTGTCG

GCCAACAGATCAGACCCTGTGACCCTGGATGTGCTGTACGGCCCTGATACCCCCATCATCTCTCCACCTGATTCTTCTTA

CCTCAGCGGAGCAAACCTGAACCTGAGCTGCCACAGCGCCAGCAACCCCAGCCCAAGTACTCTTGGCGGATCAACGGAA

TCCCTCAACAGCACACCCAAGTGCTGTTTATCGCCAAGATCACACCTAACAATAACGGCACCTACGCCTGCTTCGTGTCC

AACCTGGCTACCGGCAGAAACAACTCGATTGTTAAGAGCATCACCGTGTCTGCCAGCGGCACATCCCCTGGTCTGAGCGC

CGGCGCCACAGTCGGCATCATGATCGGCGTGCTGGTGGGCGTGGCCCTGATCCACCACCACCACCACCAC

A-CEA truncated body B fusion protein amino acid sequence SEQ ID NO: 10:

MKWVTFISLLFSSAYSKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGR

EIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLW

WVNNQSLPVSPRLQLSNGNRTLLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLS

CHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDE

DAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDP

TISPSYTYYRPGVNLSLSCHASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSA

ELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLLFNVTRNDARAYVCGIQNSVS

ANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVS

NLATGRNNSIVKSITVSASGTSPGLSAHHHHHH

The polynucleotide sequence encoding A-CEA truncated body B fusion protein SEQ ID NO: 11:

ATGAAGTGGGTGACCTTTCATCAGCCTGCTGTTCAGCTCCGCCTACTCTAAGCTGACCATCGAGTCCACCCCTTCAATGT

GGCTGAGGGCAAGGAGGTGCTGCTGCTGGTGCACAACCTGCCTCAGCACCTGTTCGGCTACAGCTGGTACAAGGGCGAGA

GAGTGGATGGCAATCGGCAGATCATCGGCTACGTGATCGGCACACAACAAGCCACCCCCGGCCCAGCCTACAGCGGACGC

GAAATCATCTACCCCAATGCCTCTCTGCTGATCCAGAACATCATTCAAAACGACACAGGCTTCTACACCCTGCACGTGAT

CAAGAGCGACCTGGTGAACGAGGAAGCCACCGGCCAGTTCAGAGTGTACCCTGAACTGCCTAAGCCCAGCATCAGCAGCA

ACAACTCCAAGCCCGTGGAAGACAAAGACGCCGTGGCCTTCACCTGCGAGCCTGAGACACAGGACGCTACATACCTGTGG

TGGGTCAACAACCAGAGCCTGCCAGTGAGCCCTAGACTGCAGCTGAGCAACGGCAACAGAACCCTGACACTGTTTAACGT

GACAAGAAATGACACCGCCAGCTACAAGTGCGAGACACAGAACCCTGTGAGCGCCAGAAGAAGCGACAGCGTGATCCTGA

ACGTGCTGTACGGCCCTGACGCCCCTACCATTTCTCCTCTGAACACCTCTTACCGGAGCGGCGAAAACCTGAACCTGAGC

TGCCATGCCGCTTCTAATCCTCCTGCTCAGTACAGCTGGTTCGTGAATGGCACCTTCCAGCAGTCTACCCAGGAGCTGTT

TATCCCCAACATCACTGTGAACAACAGCGGCAGCTATACATGTCAGGCCCACAACAGCGACACCGGACTGAACAGGACCA

CCGTAACCACAATCACCGTGTACGCCGAGCCTCCTAAGCCCTTCATCACATCTAACAACTCTAACCCTGTGGAAGATGAG

GATGCTGTTGCATTGACCTGCGAGCCTGAGATCCAGAACACCACCTACCTGTGGTGGGTGAACAACCAGAGCCTGCCCGT

GAGCCCGAGACTGCAGCTGTCTAACGACAACAGGACCCTGACCCTGCTGAGCGTGACCAGAAATGATGTGGGCCCCTACG

AGTGCGGGATCCAGAATGAGCTGAGCGTGGATCACAGCGATCCCGTGATTCTGAACGTGCTGTACGGCCCTGATGATCCA

ACCATCAGCCCTAGCTACACCTACTACAGACCAGGCGTGAACCTGAGCCTGTCTTGTCACGCCGCCAGCAATCCTCCAGC

CCAGTACAGCTGGCTGATCGACGGCAACATCCAGCAACATACCCAGGAGCTGTTCATCAGCAACATCACGGAAAAGAACT

CCGGACTTTATACCTGCCAGGCCAACAATTCTGCCAGCGGCCACTCAAGAACAACCGTCAAAAACCATCCGTGAGCGCT

GAACTGCCTAAACCCAGCATTTCTAGCAACAACTCTAAACCTGTCGAAGACAAGGACGCCGTGGCTTTTACATGTGAACC

TGAGGCCCAGAACACCACTTATCTGTGGTGGGTCAACGGCCAGAGCCTGCCCGTGTCCCCCGGCTGCAACTGTCTAATG

GCAACAGAACCCTGACACTGTTCAACGTGACACGGAACGACGCCAGAGCCTACGTTTGTGGCATCCAGAATAGCGTGAGC

GCCAACCGGAGCGACCCCGTCACCCTGGACGTGCTCTATGGCCCTGATACACCTATCATCAGCCCTCCCGACAGCAGCTA

CCTGAGCGGCGCCAATCTGAACCTGTCCTGCCACTCTGCTAGCAATCCTAGCCCACAGTACAGCTGGCGGATCAACGGAA

TCCCTCAGCAGCACACCCAGGTGCTCTTCATCGCCAAGATCACCCCTAACAACAACGGCACCTACGCTTGCTTCGTGTCC

AACCTGGCCACAGGACGGAACAACAGCATCGTGAAGTCCATCACAGTGTCCGCCTCTGGAACCAGCCCTGGCCTGAGTGC

CCACCACCACCACCACCACCAC

Embodiment 2, the expression of CEA in 293F cells

Pick the plasmids A and B prepared in step (1) of Example 1, transform the plasmids into DH5α competent cells (Tiangen), and pick a single colony for identification. Take the bacterial liquid with correct sequencing results into 100ml LB medium, culture overnight at 37°C, collect the bacterial liquid and extract the plasmid according to the instructions of the plasmid extraction kit (Qiagen). Remove endotoxin, measure the concentration, 260/280, detect endotoxin as negative, and detect the band by electrophoresis.

Use the Expi293F transfection kit to transfect 100ml of 293F cells according to the instructions, and add enhancers and adjuvants within 15-24 hours. The temperature is 36.5°C, the speed is 95rpm, and 8% CO2 is cultivated for about 4 days, and when the activity rate is higher than 60%, centrifuge at 3500rpm at 4°C for 30min to collect the supernatant.

Embodiment 3, the purification of expression product

Take 100 ml of the transient 293F cell supernatant obtained in Example 1 and Example 2, and filter it with a 0.22 μm needle filter to obtain the filtered cell supernatant. After filtration, Ni-column affinity chromatography, the protein eluted with 50mM Tris-HCl, 50mM NaCl, 200mM imidazole, pH 7.0 is the protein after elution of the target protein.

The concentration was detected by the BCA method, and the results are shown in Figure 1. Lanes 1 and 2 in Figure 1 are the stock solution expressed by carrier B and penetration, 3-8 are the eluted target proteins; lanes 9 and 10 are the stock solution expressed by carrier A and penetrate.

Calculation The target band expressed by carrier A in the supernatant is not obvious, there is almost no difference between the stock solution and the penetration, and there is no target band in the elution; the carrier B expresses the target band clearly in the supernatant, and the amount of expressed protein is 83mg/L. It can be seen that the expression level of recombinant expression of vector B in 293F cells is higher, so it can be seen that the expression level of the vector linked with the sequence encoding CEA truncation body B is higher in the 293F expression system.

Embodiment 4, flow cytometric method identification target protein activity

The antigenic activity of recombinant CEA truncation in 293F cells was detected by double-antibody sandwich method.

Sample processing before loading: After the incubation of labeled microsphere antibody and antigen, microsphere antibody-antigen complex and PE antibody, use a magnetic stand to absorb the magnet, remove the supernatant, wash the complex with a cleaning solution, and blot the waste liquid , to remove the material that is not bound to the magnetic particles, and finally wash the solution to resuspend the microsphere complex.

On-machine detection: After resuspension, the microsphere complex is detected by the flow matrix instrument. The flow matrix instrument passes the microsphere complex into a single row through the laser instrument through the sheath liquid system. The laser emits light of two wavelengths, and one beam of excitation The fluorescence of the microsphere itself is used to identify the type of microsphere, and the other is to identify the PE signal on the microsphere complex, detect the PE fluorescence signal on the corresponding microsphere, and identify the change in the concentration of the bound antigen through the difference in the PE signal.

From the results of flow cytometry, it can be seen from Table 1 that the luminescence value of carcinoembryonic antigen can reach up to 180,000, indicating that the antigen has better performance. The linear regression equation between concentration and RLU value is: y=1048.5x+3306.9, R ² =0.9966, indicating good linearity (R ² >0.99).

Table 1

Those of ordinary skill in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in practical applications, various changes can be made to it in form and details without departing from the spirit and spirit of the present invention. scope.

Claims (11)

1. A CEA truncation selected from any one of the following:

(i) A polypeptide having an amino acid sequence as shown in SEQ ID No.3 or SEQ ID No. 1;

(ii) A polypeptide having an amino acid sequence with a homology of more than 95% with the sequence shown as SEQ ID No.3 or SEQ ID No. 1.

2. An isolated polynucleotide encoding a CEA truncation, wherein the polynucleotide is codon-optimized and the polynucleotide is selected from any one of:

(i) A polynucleotide having a sequence as shown in SEQ ID NO.4 or SEQ ID NO. 2;

(ii) A polynucleotide having a homology of more than 95% with the sequence shown as SEQ ID No.4 or SEQ ID No. 2; and

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

3. An expression vector comprising the polynucleotide of claim 2.

4. The expression vector of claim 3, wherein the expression vector is a mammalian expression vector, preferably pcDNA3.4.

5. The expression vector of claim 3, wherein the expression vector comprises a polynucleotide sequence encoding an A-CEA truncation fusion protein.

6. The expression vector of claim 5, wherein the polynucleotide sequence encoding the A-CEA truncation fusion protein is selected from any one of the following:

(i) A polynucleotide having a sequence as shown in SEQ ID NO.11 or SEQ ID NO. 9;

(ii) A polynucleotide having greater than 95% homology to the sequence set forth in SEQ ID No.11 or SEQ ID No. 9; and

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

7. The expression vector of claim 3, wherein the expression vector comprises a polynucleotide sequence expressing a His x 6 tag; preferably, in the expression vector, the polynucleotide sequence expressing the His X6 tag is connected to the 3' end of the polynucleotide;

and/or, the expression vector comprises a kozak sequence located before the start codon of the polynucleotide.

8. A host cell comprising the expression vector of any one of claims 3 to 7; or

The host cell having integrated into its genome the polynucleotide of claim 2.

9. The host cell of claim 8, wherein the host cell is a CHO cell or a 293F cell.

10. A method for preparing a CEA truncation, comprising the steps of:

transforming a host cell with a vector comprising the polynucleotide of claim 2;

culturing the host cell to express the CEA truncation.

11. A kit, comprising: the polynucleotide of claim 2; or

The expression vector of any one of claims 3 to 7; or alternatively

The host cell of claim 8 or 9; or alternatively

The CEA truncation of claim 1.

Images