Preparation method and application of claudin2-magea3 fusion tumor antigen protein

Abstract

The application provides a preparation method and application of a Claudin2-MAGEA3 fusion tumor antigen protein, the preparation method comprising the following steps: screening antigen fragments with strong antigenicity in Claudin2 protein and MAGEA3 protein respectively, and chemically synthesizing a coding gene of the Claudin2 antigen fragment and the MAGEA3 antigen fragment in series, and preparing a fusion protein, which is used for self-antibody detection, improves the detection rate of anti-Claudin2 antibodies and anti-MAGEA3 antibodies in tumor patient samples, can efficiently predict whether an individual is a lung cancer patient, has the advantages of high sensitivity and high specificity, reduces the occurrence of clinical missed detection, and has important scientific significance and clinical application value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering technology and diagnostic reagents, specifically to a method for preparing a Claudin2-MAGEA3 fusion tumor antigen protein and its application. Background Technology

[0002] Lung cancer is one of the most common malignant tumors in the world, and its incidence and mortality rates are increasing year by year. Currently, it has the highest incidence rate in the world, seriously threatening human health and life. Lung cancer is a disease that tends to be insidious, often only showing clinical symptoms in the late stages of the disease. 70% to 80% of lung cancer patients are diagnosed with lung cancer symptoms at the middle or late stages, when cancer cells have already spread, missing the best time for treatment, and resulting in a low five-year survival rate.

[0003] Autoantibodies are antibodies against the body's own tissues, organs, cells, and cellular components. In the early stages of cancer development, exposure to tumor-associated antigens can be recognized by the body's immune system, leading to the production of tumor-associated autoantibodies. High levels of these autoantibodies can even be maintained in peripheral blood and can be sensitively detected by conventional techniques in the field, such as enzyme-linked immunosorbent assays (ELISA). It is widely recognized in the field that autoantibodies produced by tumor antigens are a good indicator for early cancer diagnosis. Utilizing tumor-induced autoantibodies to reflect the development and progression of cancer in patients is becoming an important direction for finding new targets for early cancer diagnosis and prognosis.

[0004] However, due to tumor heterogeneity and differences in immune system responses among individuals, the sensitivity of a single tumor autoantibody in cancer patients is insufficient (typically 5%–20%). Therefore, a single autoantibody biomarker is inadequate to provide information on the occurrence and progression of lung cancer. Combining multiple different tumor autoantibodies can increase detection sensitivity, but this method has drawbacks such as numerous steps, high cost, low efficiency, insufficient specificity, and long detection time. Therefore, there is an urgent need to provide a method that can improve the detection rate and increase the sensitivity of lung cancer autoantibodies.

[0005] Claudin2 and MAGEA3 are two tumor autoantigens associated with lung cancer. Claudin2, as a major component of the tight junction complex, regulates the permeability of epithelial cells; while in lung adenocarcinoma tissue, claudin2 accelerates cancer cell proliferation and metastasis. Multiple studies inhibiting claudin2 activity or expression have shown that reducing claudin2 function can significantly inhibit cancer cell growth. MAGEA3, also known as tumor testis antigen 1.3, is expressed in nearly half of stage II non-small cell lung cancer tissues and has shown potential as a prognostic marker. It is also currently undergoing clinical trials as a target for tumor immunotherapy. Using either of these as antigen molecules for lung cancer autoantibody detection results in a certain rate of false negatives. Therefore, it is necessary to prepare epitopes that fuse the two antigens to develop fusion antigens to achieve highly sensitive and specific early detection of lung cancer. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a fusion protein for screening early-stage lung cancer. By screening antigen fragments with strong antigenic determinants in Claudin2 and MAGEA3 proteins respectively, and chemically synthesizing the coding genes of the Claudin2 and MAGEA3 antigen fragments in tandem, a fusion protein is prepared for autoantibody detection. This can efficiently predict whether an individual has lung cancer, improve the specificity and sensitivity of early lung cancer screening, and reduce the occurrence of clinical missed detections. It has significant scientific and clinical application value.

[0007] On one hand, the present invention provides a fusion protein comprising a Claudin2 antigen fragment, a MAGEA3 antigen fragment, and a linker peptide connecting the Claudin2 antigen fragment and the MAGEA3 antigen fragment.

[0008] This invention integrates a large amount of public data, compares autoantibodies in the serum of lung cancer patients and normal people, screens and obtains a series of antigen proteins that can better distinguish between lung cancer patients and normal people, and further screens out Claudin2 antigen protein and MAGEA3 antigen protein that can be used to combine and prepare fusion proteins.

[0009] The Claudin2 antigen fragment is an antigen fragment selected from the amino acid sequence of the Claudin2 protein that can elicit the strongest antigenic response; the MAGEA3 antigen fragment is an antigen fragment selected from the amino acid sequence of the MAGEA3 protein that can elicit the strongest antigenic response.

[0010] The Claudin2 protein has the amino acid sequence shown in Sequence Listing Seq ID NO.5, and the MAGEA3 protein has the amino acid sequence shown in Sequence Listing Seq ID NO.6.

[0011] This invention uses a fusion protein prepared from Claudin2 antigen fragment and MAGEA3 antigen fragment as a biomarker for lung cancer detection. The detection rate of lung cancer patients is significantly higher than that of the single protein.

[0012] Furthermore, the Claudin2 antigen fragment has the amino acid sequence shown in Sequence Listing Seq ID NO.1; the MAGEA3 antigen fragment has the amino acid sequence shown in Sequence Listing Seq ID NO.2.

[0013] The Claudin2 protein fragment is amino acids 184 to 194 of the Claudin2 protein, and the MAGEA3 protein fragment is amino acids 1 to 112 of the MAGEA3 protein.

[0014] Furthermore, the Claudin2 antigen fragment is located at the N-terminus of the fusion protein, and the MAGEA3 antigen fragment is located at the C-terminus of the fusion protein; the amino acid sequence of the linker peptide is glycine-glycine-serine.

[0015] The Claudin2 protein fragment is located at the N-terminus (amino terminus) of the fusion protein, and the MAGEA3 protein fragment is located at the C-terminus (carboxyl terminus) of the fusion protein.

[0016] Furthermore, the fusion protein has an amino acid sequence as shown in Sequence Listing Seq ID NO.3.

[0017] On the other hand, the present invention provides a gene encoding a fusion protein, the gene being capable of encoding the fusion protein as described above.

[0018] Furthermore, the gene has a nucleotide sequence as shown in Sequence Listing Seq ID NO.4.

[0019] In some embodiments, the gene sequence has two restriction endonucleases, Ncol and Ndel, at both ends.

[0020] In another aspect, the present invention provides a kit for predicting whether an individual is a lung cancer patient, the kit comprising the fusion protein as described above, or the fusion protein encoded by the coding gene as described above.

[0021] In another aspect, the present invention provides the use of the fusion protein described above for preparing reagents to predict whether an individual is a lung cancer patient.

[0022] Furthermore, the present invention provides a method for preparing the fusion protein as described above, the method comprising the following steps:

[0023] (1) Screening out Claudin2 antigen fragments containing strong antigenic determinants in Claudin2 antigen protein, and screening out MAGEA3 antigen fragments containing strong antigenic determinants in MAGEA3 antigen protein.

[0024] (2) Chemically synthesize a coding gene for a tandem Claudin2 antigen fragment and a MAGEA3 antigen fragment, wherein the coding gene has a nucleotide sequence as shown in Seq ID NO.4 of the sequence listing;

[0025] (3) Construct recombinant plasmids containing coding genes;

[0026] (4) Transform Escherichia coli with recombinant plasmid and select positive expression bacteria.

[0027] In some methods, step (1) uses an online analysis tool to screen for Claudin2 protein fragments (Claudin2 protein contains amino acids 184 to 194 of a strong antigenic determinant) and MAGEA3 protein fragments (MAGEA3 protein contains amino acids 1 to 112 of a strong antigenic determinant).

[0028] Further, step (3) is as follows: extract PGEM-5zf, digest it with Ncol and Ndel, and recover the digested plasmid fragments by electrophoresis; digest the chemically synthesized coding gene with Ncol and Ndel, and recover the digested gene fragments by electrophoresis; ligate the digested plasmid fragments and the digested gene fragments at a ratio of 1:3-10 using T4 ligase.

[0029] In some methods, PGEM-5zf is extracted, double-digested with Ncol and Ndel, and the double-digested plasmid fragments are recovered by gel electrophoresis; the chemically synthesized fusion protein gene fragment is double-digested with Ncol and Ndel, the double-digested gene fragments are recovered by electrophoresis, and stored at -20℃; the double-digested plasmid fragments and double-digested gene fragments are ligated at a ratio of 1:3-10 using T4 ligase overnight at 16℃, and the resulting recombinant plasmid is PGEM-5zf-Claudin2+MAGEA3.

[0030] Further, step (4) is as follows: transform the recombinant plasmid into Escherichia coli BL21(DE3) and spread it on an LB plate containing ampicillin and incubate overnight in a constant temperature incubator; the next day, randomly pick the transformed colonies, extract the plasmid, and digest it with Ndel and Ncol enzymes respectively. After digestion, run electrophoresis and see the corresponding target fragment and vector. The expression vector is successfully constructed, which is the positive expression bacteria.

[0031] In some methods, the recombinant plasmid is transformed into Escherichia coli BL21(DE3), plated on LB agar plates containing ampicillin (60 ug / ml), and incubated overnight at 37°C. The next day, transformed colonies and control colonies (plasmid-transformed bacteria PGEM-5zf) are randomly selected, and the plasmids are extracted and digested with Ndel and Ncol enzymes, respectively. After digestion, electrophoresis is performed, and the corresponding target fragment and vector can be seen in both cases. The expression vector is successfully constructed, and the bacteria are positive expression bacteria.

[0032] Furthermore, the preparation method further includes step (5): expressing the target protein by positive expression bacteria.

[0033] The above-mentioned positive expression bacteria were inoculated into test tubes containing 2 ml LB medium (60 μg / ml ampicillin) and shaken at 37°C for 4 h. IPTG was added to a final concentration of 0.5 mmol / L, and induction was continued at 37°C for another 6 h. The bacterial precipitate was collected by centrifugation, and the precipitate was broken up. The supernatant and precipitate were analyzed by SDS-PAGE, and the expressed target protein was obtained from the supernatant.

[0034] Furthermore, the preparation method also includes step (6): affinity chromatography column separation and purification.

[0035] Select single colonies of engineered bacteria that efficiently express the recombinant protein and inoculate them into Erlenmeyer flasks containing 100 ml of LB liquid medium. Add ampicillin to a final concentration of 60 μg / ml and incubate overnight at 37°C in a shaker. The next day, inoculate the bacterial culture and LB medium at a ratio of 1:10 into Erlenmeyer flasks containing 1000 ml of LB (60 μg / ml ampicillin) medium and incubate for 4 h. Add IPTG (final concentration 0.5 mmol / L) and continue induction at 37°C for 6 h. Centrifuge 1000 ml of engineered bacteria that have been induced to express the fusion protein at high speed (10000 rpm) and low temperature (4°C) for 10 min. Resuspend the precipitated bacterial cells in 1 / 10 of the original centrifugation volume of lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 15 mM NaCl, 10 mM DTT), sonicate the cells on ice, and centrifuge at high speed (12000 rpm) and low temperature (4°C) for 30 min. Collect the supernatant of the bacterial culture.

[0036] After equilibrating the nickel ion affinity chromatography column with equilibration buffer (20 mM PB, pH 7.4), the supernatant of the bacterial culture collected by sonication was directly loaded onto the column at a flow rate of 1.5 ml / min. After loading, the column was washed with equilibration buffer, and the proteins were eluted sequentially with elution buffers containing 20, 50, 100, 200, and 500 mM imidazole. The proteins in each elution peak were collected, and the proteins in each peak were detected by SDS-PAGE electrophoresis.

[0037] In another aspect, the present invention provides a system for predicting whether an individual is a lung cancer patient, the system including a data analysis module; the data analysis module is used to analyze the detection of biomarkers, the biomarkers being autoantibodies of the fusion protein as described above.

[0038] The biomarker described in this invention is an autoantibody, and more specifically, the autoantibody is an anti-Claudin2 antibody and an anti-MAGEA3 antibody.

[0039] Furthermore, the system also includes a biomarker detection module and a data input / output interface; the biomarker detection module is used to detect the content of autoantibodies in serum through fusion protein to obtain detection values; the data input interface is used to input the autoantibody detection values ​​of the fusion protein, and after analysis by the data analysis module, the data output interface is used to output the screening results for lung cancer.

[0040] Furthermore, the analysis method of the data analysis module is as follows: using the fusion protein as an antigen, the presence of autoantibodies of Claudin2 and / or MAGEA3 above the threshold in the serum is detected. If they are present, the patient is identified as a potential lung cancer patient; if they are not present, the patient is identified as a normal person.

[0041] The fusion protein provided by this invention for predicting whether an individual is a lung cancer patient has the following beneficial effects:

[0042] 1. Claudin2 and MAGEA3 proteins were selected, and a fusion protein was prepared using in vitro recombinant protein expression technology. The detection rate of this fusion protein in screening for early lung cancer was significantly higher than that of the single protein.

[0043] 2. The selected Claudin2 and MAGEA3 protein fragments are highly antigenic. By linking the two highly antigenic fragments with glycine-glycine-serine, the detection rate of anti-Claudin2 and anti-MAGEA3 antibodies in tumor patient samples was improved, and the occurrence of clinical missed detection was reduced.

[0044] 3. It can efficiently predict whether an individual is a lung cancer patient, and has the advantages of high sensitivity and high specificity, which has important scientific significance and clinical application value.

[0045] Detailed description

[0046] (1) Screening or testing

[0047] Screening or testing here refers to detecting or analyzing biomarkers in a sample, or determining the content of a target biomarker, such as its absolute or relative content. The presence or quantity of the target biomarker then indicates whether the individual providing the sample may have or suffer from a certain disease, or the likelihood of having a certain disease. The meanings of screening and testing are interchangeable here. The results of such testing or screening cannot be directly used as a direct result of disease; rather, they are intermediate results. To obtain a direct result, further auxiliary methods such as pathology or anatomy are needed to confirm the presence of a specific disease. For example, this invention provides biomarkers that are correlated with whether an individual has lung cancer; changes in the levels of these biomarkers are directly correlated with whether an individual has lung cancer.

[0048] (2) Effective association between biomarkers or markers and whether an individual has lung cancer.

[0049] In this invention, biomarkers and biomarkers have the same meaning. Here, "related" refers to a direct correlation between the presence or change in the concentration of a biomarker in a sample and the efficacy of a specific treatment method. For example, a relative increase or decrease in concentration indicates a higher or lower likelihood that the treatment method will have a beneficial effect.

[0050] The presence or absence, or the increase or decrease of the content of the fusion protein autoantibody biomarker for lung cancer patients provided by this invention, is directly related to whether the individual is a lung cancer patient.

[0051] (3) In this invention, the terms "antigen" and "antigen protein" are used interchangeably. The terms "antibody" and "autoantibody" are used interchangeably in this invention. Attached Figure Description

[0052] Figure 1 The ROC curve for the antigen protein Claudin2 used in lung cancer diagnosis in Example 1 is shown.

[0053] Figure 2 The ROC curve for the antigen protein MAGEA3 used in Example 1 for lung cancer diagnosis is shown.

[0054] Figure 3 The ROC curve of the antigen protein RALGDS used for lung cancer diagnosis in Example 1 is shown.

[0055] Figure 4 The ROC curve for the antigen protein KRT8 used in Example 1 for lung cancer diagnosis is shown.

[0056] Figure 5 The ROC curve for the antigen protein AIF1 used in lung cancer diagnosis in Example 1 is shown.

[0057] Figure 6 The ROC curve for the combination of antigen proteins Claudin2 and MAGEA3 in Example 1 for lung cancer diagnosis is shown.

[0058] Figure 7 This is the preliminary screening result of the Claudin2 protein fragment in Example 2;

[0059] Figure 8 This is a scatter plot showing the relationship between different fragments of the Claudin2 protein used in clinical samples for the detection and evaluation of autoantibody content.

[0060] Figure 9 This is the preliminary screening result of the MAGEA3 protein fragment in Example 2;

[0061] Figure 10 This is a scatter plot showing the relationship between different fragments of the MAGEA3 protein used in Example 2 for the detection and evaluation of autoantibody content in clinical samples.

[0062] Figure 11 This is a graph showing the results of detecting serum from lung cancer patients and normal human serum using the fusion protein in Example 4.

[0063] Figure 12 The image shows the results of detecting clinical samples using the fusion protein and the independent claudin2 and MAGEA3 proteins in Example 4. Detailed Implementation

[0064] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The reagents used in this embodiment are all known products, and unless otherwise specified, they are all commercially available products.

[0065] Example 1: Screening of antigen proteins

[0066] This embodiment summarizes and synthesizes 169 lung cancer-related antigen proteins from a large amount of public data. Autoantibodies against purified antigen proteins were detected in the serum of 47 patients diagnosed with lung cancer and 47 healthy individuals to identify autoantibody biomarkers more relevant to lung cancer screening. After initial screening (identifying positively and negatively correlated antigens with good and poor predictive performance in lung cancer diagnosis), 20 autoantibody antigen proteins were identified, as shown in Table 1. These 20 autoantibodies showed significant correlation with determining whether an individual has lung cancer. The Uniprot database can be accessed at www.uniprot.org.

[0067] Table 1. Antigen proteins of 20 autoantibodies obtained in the initial screening

[0068] antigen protein Uniprot database serial number antigen protein Uniprot database serial number CIP2A Q8TCG1 TXNDC2 Q86VQ3 CTAG2 O75638 RASSF7 Q02833 KRT8 P05787 LIN28B Q6ZN17 SS18 Q15532 Claudin2 P57739 NPM1 P06748 Livin-1 Q96CA5 MAGEB1 P43366 MAGEA3 P43357 CDK2 P24941 BARD1 Q99728 PBRM1 Q86U86 PAGE3 Q5JUK9 RALGDS Q12967 CT47A Q5JQC4 Trim21 P19474 AIF1 P55008

[0069] Serum samples from 47 lung cancer patients and 47 healthy controls were analyzed to detect 20 candidate autoantibody molecules. The diagnostic performance of these antigen proteins for lung cancer was evaluated according to RECIST v1.1 (Response Evaluation Criteria in Solid Tumors). The levels of the 20 candidate autoantibodies in the serum of lung cancer patients and healthy controls were measured using enzyme-linked immunosorbent assay (ELISA). OddO values ​​were used to plot a scatter plot of the relationship between the level of each autoantibody and whether the serum indicated lung cancer. The results showed that 5 of the 20 candidate autoantibody molecules exhibited significantly higher OD values ​​in the serum of lung cancer patients than in the serum of healthy controls (the difference was highly significant); while the differences for the remaining 15 autoantibodies were significantly less pronounced than those of the 5 autoantibodies.

[0070] The antigenic proteins of these five autoantibodies are Claudin2, MAGEA3, RALGDS, KRT8, and AIF1 antigenic proteins. Their sensitivity, specificity, and AUC values ​​for diagnosing lung cancer are shown in Table 1, and the ROC curves are shown in [Table 1]. Figures 1-5 .

[0071] Table 1. Diagnostic performance of the five selected antigen proteins for lung cancer

[0072] Serial Number antigen protein Sensitivity (%) Specificity (%) AUC value 1 Claudin2 25.00 87.23 0.5191 2 MAGEA3 21.28 91.49 0.5310 3 RALGDS 28.26 88.37 0.5657 4 KRT8 17.02 85.11 0.5109 5 AIF1 26.00 85.00 0.6053

[0073] To prepare the fusion protein, this embodiment also combined the five screened antigen proteins in pairs, selecting the two most suitable antigen proteins for preparing a fusion protein with high diagnostic performance for early lung cancer. The sensitivity, specificity, and AUC values ​​of the five antigen proteins after pairwise combinations for diagnosing lung cancer are shown in Table 2.

[0074] Table 2. Diagnostic efficacy of lung cancer after pairwise combinations of the five antigen proteins.

[0075] Serial Number Antigen protein combination Sensitivity (%) Specificity (%) AUC value 1 Claudin2+MAGEA3 40.43 89.36 0.7082 2 Claudin2+RALGDS 32.72 89.14 0.6837 3 Claudin2+KRT8 36.15 90.28 0.6923 4 Claudin2+AIF1 37.22 86.73 0.6752 5 MAGEA3+AIF1 31.54 87.38 0.6107 6 MAGEA3+RALGDS 36.26 88.69 0.6385 7 MAGEA3+KRT8 29.37 84.01 0.6298 8 RALGDS+KRT8 33.28 88.34 0.6853 9 RALGDS+AIF1 32.15 86.77 0.6324 10 KRT8+AIF1 35.86 85.21 0.6197

[0076] As shown in Table 2, the combination of Claudin2 antigen protein and MAGEA3 antigen protein exhibits the best diagnostic performance for lung cancer, with a sensitivity of 40.43%, a specificity of 89.36%, and an AUC value of 0.7082. The ROC curve is shown below. Figure 6As shown. Therefore, it is preferable to use a combination of Claudin2 antigen protein and MAGEA3 antigen protein for the preparation of fusion proteins.

[0077] Example 2: Screening of Claudin2 and MAGEA3 antigen fragments

[0078] This embodiment utilizes online analysis tools such as ExPasy, TMHMM, and SignalIP 4.1 to screen for fragments with strong antigenic determinants in the Claudin2 and MAGEA3 protein fragments. The screening results are shown in […]. Figure 7 and 9 ,in Figure 7 Preliminary screening results for Claudin2 protein fragments; Figure 9 Preliminary screening results for MAGEA3 protein fragments. Figure 7 It can be roughly observed that the segment with a strong antigenic determinant in the Claudin2 protein is likely located between amino acids 184 and 220. Figure 9 It can be roughly seen that the fragments with strong antigenic determinants in the MAGEA3 protein are likely located between the 1st and 105th amino acids.

[0079] Simultaneously, in this embodiment, different fragments of Claudin2 and MAGEA3 proteins were prepared (the preparation method was the same as in Example 3: first, the coding gene was synthesized, then a recombinant plasmid containing the coding gene was constructed, transformed into E. coli, and positive expression bacteria were selected to express the protein). These fragments were used to detect autoantibodies in the serum of 47 lung cancer patients and 47 normal individuals (the detection method was the same as in Example 4, using an indirect enzyme-linked immunosorbent assay). The OD values ​​of the autoantibodies were detected, and the results are as follows: Figure 8 and Figure 10 As shown, where Figure 8 A scatter plot showing the relationship between different fragments of the Claudin2 protein used to evaluate the content of autoantibodies in clinical samples. Figure 10 A scatter plot showing the relationship between different fragments of the MAGEA3 protein used to evaluate the content of autoantibodies in clinical samples.

[0080] according to Figure 8 It can be seen that the fragment located between amino acids 184 and 194 in the Claudin2 protein fragment is the fragment that can elicit a stronger antigen response, so this fragment is preferred for preparing the fusion protein.

[0081] according to Figure 10 It can be seen that the fragment from amino acid 1 to amino acid 112 in the MAGEA3 protein fragment is the fragment that can elicit a stronger antigen response, so this fragment is preferred for preparing the fusion protein.

[0082] Example 3: Preparation of Fusion Protein

[0083] The fusion protein prepared in this embodiment is a fusion protein containing Claudin2 and MAGEA3 protein fragments, linked by a connecting peptide. The Claudin2 protein fragment is located at the amino terminus of the fusion protein, and the MAGEA3 protein fragment is located at the carboxyl terminus. The Claudin2 and MAGEA3 protein fragments are linked by a glycine-glycine-serine residue. The Claudin2 protein fragment consists of amino acids 184 to 194 (Seq ID NO. 1), and the MAGEA3 protein fragment consists of amino acids 1 to 112 (Seq ID NO. 2). The amino acid sequence of the connecting peptide is glycine-glycine-serine. The amino acid sequence of this fusion protein is shown in Seq ID NO. 3, and the encoding gene sequence is shown in Seq ID NO. 4. The specific preparation method is as follows:

[0084] (1) The coding gene sequence of the fusion protein (Seq ID NO.4) was chemically synthesized using DNA solid-phase synthesis.

[0085] (2) Construction of recombinant plasmids expressing Claudin2 and MAGEA3 protein fragments

[0086] PGEM-5zf was extracted and double-digested with Ncol and Ndel. The double-digested plasmid fragments were recovered by gel electrophoresis. The chemically synthesized autoantigen fusion protein gene fragment was double-digested with Ncol and Ndel. The double-digested gene fragments were recovered by electrophoresis and stored at -20℃. The double-digested plasmid fragments and double-digested gene fragments were ligated at a ratio of 1:3-10 using T4 ligase overnight at 16℃. The resulting recombinant plasmid was PGEM-5zf-Claudin2+MAGEA3.

[0087] (3) Screening and identification of recombinant plasmids

[0088] The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and plated on LB agar plates containing ampicillin (60 μg / ml). The plates were incubated overnight at 37°C. The next day, transformed colonies and control colonies (transformed with plasmid PGEM-5zf) were randomly selected, and the plasmids were extracted and digested with Ndel and Ncol enzymes, respectively. After digestion, electrophoresis showed that the corresponding target fragment and vector were visible in both cases, indicating successful construction of the expression vector. These were the positive expression bacteria.

[0089] (4) High-efficiency expression of recombinant protein by engineered bacteria

[0090] The positive expression bacteria were inoculated into test tubes containing 2 ml LB medium (60 μg / ml ampicillin) and shaken at 37°C for 4 h. IPTG was added to a final concentration of 0.5 mmol / L, and induction was continued at 37°C for another 6 h. The bacterial precipitate was collected by centrifugation, and the precipitate was broken up. The supernatant and precipitate were analyzed by SDS-PAGE, and the expressed target protein was obtained from the supernatant.

[0091] (5) Purification of the expressed protein

[0092] Single colonies of engineered bacteria expressing the recombinant protein with high efficiency were selected and inoculated into Erlenmeyer flasks containing 100 ml of LB liquid medium. Ampicillin was added to a final concentration of 60 μg / ml, and the flasks were incubated overnight at 37°C in a shaker. The next day, the bacterial culture was inoculated into Erlenmeyer flasks containing 1000 ml of LB (60 μg / ml ampicillin) medium at a ratio of 1:10. After incubation for 4 h, IPTG (final concentration 0.5 mmol / L) was added, and the culture was induced for another 6 h at 37°C. The 1000 ml of engineered bacteria expressing the fusion protein was centrifuged at high speed (10000 rpm) and low temperature (4°C) for 10 min. The precipitated bacterial cells were resuspended in 1 / 10 of the original centrifugation volume of lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 15 mM NaCl, 10 mM DTT), and the cells were sonicated on ice. The cells were then centrifuged at high speed (12000 rpm) and low temperature (4°C) for 30 min, and the supernatant was collected.

[0093] After equilibrating the nickel ion affinity chromatography column with equilibration buffer (20 mM B, pH 7.4), the supernatant of the bacterial culture collected by sonication was directly loaded onto the column at a flow rate of 1.5 ml / min. After loading, the column was washed with equilibration buffer, and proteins were eluted sequentially with elution buffers containing 20, 50, 100, 200, and 500 mM imidazole. The proteins in each elution peak were collected, and SDS-PAGE electrophoresis was used to detect the proteins in each peak. The fusion protein was obtained with a purity of 90%.

[0094] Example 4: Detection performance of fusion protein

[0095] In this embodiment, the fusion protein purified in Example 3 was diluted with carbonate buffer (50 mM, pH 9.6) and coated onto ELISA plates at 100 μL / well and 100 ng / well, and incubated overnight at 4°C. After washing, 200 μL / well of 10% fetal bovine serum PBS was added and the plates were blocked at 37°C for 2 hours. After washing, the plates were stored at 4°C for later use.

[0096] A fusion protein indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the reactivity of autoantibodies in lung cancer patients' and healthy individuals' serum. Specifically, the method involved detecting the reactivity of autoantibodies in the serum of lung cancer patients and healthy individuals using indirect ELISA. Serum or plasma samples were diluted 1:110 with phosphate buffer and added to microwells (50 mL / well). After washing with washing buffer to remove unbound serum or plasma components, horseradish peroxidase (HRP)-conjugated anti-human IgG was added to each well for reaction. Then, the reaction substrate TMB (3,3',5,5'-tetramethylbenzidine) was added for color development. Stop solution (1N HCl) was added, and the absorbance was measured at 450 nm using a microplate reader (OD). Results are as follows: Figure 11 As shown. By Figure 11 The results showed that the fusion protein exhibited strong immunogenicity in the serum of lung cancer patients but did not react with the serum of normal individuals, indicating that the fusion protein has good antigenicity and specificity.

[0097] Then, this fusion antigen was compared with the independent claudin2 and MAGEA3 proteins, and the results were as follows: Figure 12 As shown, the immunogenicity of the fusion protein is significantly stronger than that of the individual protein molecules, demonstrating that the fusion protein has better sensitivity in lung cancer detection. The test results showed that its sensitivity reached 55.65%, its specificity reached 86.37%, and its AUC value reached 0.7533 (which is a further improvement compared to the combination of full-length claudin2 and MAGEA3 (0.7082)).

[0098] This patent is not limited to the specific implementation methods described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this patent.

[0099] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

[0100] sequence list

[0101] Seq ID NO.1

[0102] Claudin2 antigen fragment:

[0103] SCSSQRNRSNY Seq ID NO.2

[0104] MAGEA3 antigen fragment:

[0105] MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGAS

[0106] SLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRK Seq ID NO.3

[0107] Amino acid sequence of the fusion protein:

[0108] MGSSSCSSQRNRSNYAARGGGGSGGGGSGGGGSCSSQRNRSNYAARGSGSGSGSHHHHHHEQKLISE

[0109] EDLGSMPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQS

[0110] PQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKSeq ID NO.4

[0111] Gene sequence of the fusion protein:

[0112] ATGGGCAGCAGCAGCTGTTCTTCTCAGCGTAATCGTTCTAATTATGCTGCTCGTGGTGGTGGCGGTT

[0113] CTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTTGTTCTTCTCAGCGTAATCGTTCTAACTATGCTGC

[0114] TCGTGGCAGTGGAAGTGGTTCCGGGAGTCATCATCATCATCATCACGAACAAAAACTCATCTCAGA

[0115] AGAGGATCTGGGATCCATGCCTCTTGAGCAGAGGAGTCAGCACTGCAAGCCTGAAGAAGGCCTTG

[0116] AGGCCCGAGGAGAGGCCCTGGGCCTGGTGGGTGCGCAGGCTCCTGCTACTGAGGAGCAGGAGGC

[0117] TGCCTCCTCCTCTTCTACTCTAGTTGAAGTCACCCTGGGGGAGGTGCCTGCTGCCGAGTCACCAGA

[0118] TCCTCCCCAGAGTCCTCAGGGAGCCTCCAGCCTCCCCACTACCATGAACTACCCTCTCTGGAGCCA

[0119] ATCCTATGAGGACTCCAGCAACCAAGAAGAGGAGGGGCCAAGCACCTTCCCTGACCTGGAGTCCG

[0120] AGTTCCAAGCAGCACTCAGTAGGAAG

[0121] Seq ID NO.5

[0122] Claudin 2:

[0123] MASLGLQLVGYILGLLGLLGTLVAMLLPSWKTSSYVGASIVTAVGFSKGLWMECATHSTGITQCDIYST

[0124] LLGLPADIQAAQAMMVTSSAISSLACIISVVGMRCTVFCQESRAKDRVAVAGGVFFILGGLLGFIPVAW

[0125] NLHGILRDFYSPLVPDSMKFEIGEALYLGIISSLFSLIAGIILCFSCSSQRNRSNYYDAYQAQPLATRSSPR

[0126] PGQPPKVKSEFNSYSLTGYV

[0127] Seq ID NO.6

[0128] MAGEA3:

[0129] MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGAS

[0130] SLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLG

[0131] SVVGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAI

[0132] IAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPR

[0133] ALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEE。

Claims

1. A fusion protein, characterized in that, It includes a Claudin2 antigen fragment, a MAGEA3 antigen fragment, and a linker peptide connecting the Claudin2 antigen fragment and the MAGEA3 antigen fragment; the amino acid sequence of the fusion protein is shown in Sequence Listing SeqID NO.

3.

2. A gene encoding a fusion protein, characterized in that, It can encode the fusion protein as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence is shown in Sequence Listing Seq ID NO.

4.

4. A kit for predicting whether an individual has lung cancer, characterized in that, This includes the fusion protein as described in claim 1, or the fusion protein encoded by the coding gene as described in claim 2 or 3.

5. The use of the fusion protein of claim 1 for preparing a reagent to predict whether an individual is a lung cancer patient.

6. The method for preparing the fusion protein as described in claim 1, characterized in that, Includes the following steps: (1) Screen out Claudin2 antigen fragments containing strong antigenic determinants in Claudin2 antigen protein, and screen out MAGEA3 antigen fragments containing strong antigenic determinants in MAGEA3 antigen protein. (2) The coding gene of the Claudin2 antigen fragment and the MAGEA3 antigen fragment tandemly synthesized by chemical synthesis, wherein the nucleotide sequence of the coding gene is shown in Seq ID NO.4 of the sequence listing; (3) Construct recombinant plasmids containing coding genes; (4) Transform Escherichia coli with recombinant plasmid and select positive expression bacteria.

7. The preparation method according to claim 6, characterized in that, Step (3) is as follows: extract PGEM-5zf, digest it with Ncol and Ndel, and recover the digested plasmid fragments by electrophoresis; digest the chemically synthesized coding gene with Ncol and Ndel, and recover the digested gene fragments by electrophoresis; ligate the digested plasmid fragments and the digested gene fragments at a ratio of 1:3-10 using T4 ligase.

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