Recombinant protein G3P1-12, and preparation method and application thereof

Abstract

This invention discloses a recombinant protein G3P1-12, its preparation method, and its applications, belonging to the field of DNA recombination technology in bioengineering. This invention utilizes phage display technology to clone a gene fragment encoding the N-terminal 1-12 peptide segment of the P53 protein into the PIII protein gene of a filamentous phage vector, preparing a recombinant protein displaying the P53 protein epitope. This recombinant protein is then displayed on the surface of phages for the detection of p53 antibodies in the serum of cancer patients. This invention also discloses the application of recombinant protein G3P1-12 in the preparation of a biological product for detecting p53 antibodies in the serum of cancer patients. This biological product has advantages such as high specificity, high sensitivity, simple preparation, and low cost.

Description

Technical Field

[0001] This invention relates to the field of DNA recombination technology in bioengineering, and more specifically to a recombinant protein G3P1-12, its preparation method, and its applications. Background Technology

[0002] Cancer is a major killer threatening human health, with approximately 2.6 million new cases and 1.8 million deaths annually in my country. Many factors contribute to this phenomenon, one important reason being the current inability to effectively diagnose the development and progression of tumors, especially in their early stages.

[0003] p53 is a tumor suppressor gene whose protein plays an important regulatory role in maintaining normal cell division and growth. However, once this gene mutates, the mutant protein it encodes has a prolonged half-life, loses its tumor suppressor function, accumulates in cells, and stimulates the immune system to produce p53 antibodies. Studies have shown that serum p53 antibodies can serve as a broad-spectrum tumor marker for early tumor detection and screening of high-risk groups for cancer.

[0004] Currently, the detection of serum p53 antibodies is mainly based on recombinant p53 protein. However, the preparation and purification of this protein are cumbersome, time-consuming, relatively expensive, and have low sensitivity. Therefore, providing a recombinant protein G3P1-12, its preparation method, and its applications is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a recombinant protein G3P1-12, its preparation method and application. The preparation method is simple, low-cost and highly sensitive. The recombinant protein G3P1-12 can be used for the detection of p53 antibodies in the serum of tumor patients.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The epitopes recognized by serum P53 antibodies in cancer patients are mainly located in the N segment of the P53 protein, where amino acids 1-12 of the N segment are a key epitope for antibody recognition.

[0008] A recombinant protein G3P1-12 is composed of the P53 protein epitope (MEEPQSDPSVEP) and the filamentous phage minor coat protein g3p. Specifically, the amino acid sequence MEEPQSDPSVEP, representing amino acids 1-12 of the N segment of the P53 protein, is displayed on the filamentous phage minor coat protein PIII. The amino acid sequence of the recombinant protein G3P1-12 is shown in SEQ ID NO.1. The bolded black portion represents the P53 protein epitope (MEEPQSDPSVEP).

[0009]

[0010] Furthermore, the nucleotide sequence encoding the recombinant protein G3P1-12 is shown in SEQ ID NO.2.

[0011] The bolded portion (nucleotide sequence: 67-102) is the nucleotide sequence encoding the P53 protein epitope (MEEPQSDPSVEP); the nucleotide sequence before the bolded portion (nucleotide sequence: 1-66) is the signal peptide encoding the PIII protein; the bolded and underlined portion (nucleotide sequence 103-126) is the linker, which connects to the PIII protein of the target peptide polyphage, with the purpose of enabling the displayed exogenous peptide to better perform its function; the nucleotide sequence after the bolded and underlined portion (127-1344) is the gene of the PIII protein.

[0012]

[0013]

[0014] Furthermore, a method for preparing recombinant protein G3P1-12 includes the following specific steps:

[0015] (1) The vector fADL-le was digested with BglI enzyme and the digested vector fADL-le was recovered;

[0016] (2) Synthetic phage display epitope MEEPQSDPSVEP:

[0017] Two complementary DNA fragments encoding the N-terminal amino acids MEEPQSDPSVEP of the P53 protein were synthesized.

[0018] 5'-CGGCCATGGCAATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCGGCCCGGG-3'; SEQ ID NO.3;

[0019] 5'-GGCCGGGCTCGACGCTAGGATCTGACTGCGGCTCCCTCCATTGCCATGGCCGGCT-3'; SEQ ID NO.4;

[0020] The two fragments were dissolved and mixed in equimolar amounts. The mixture was denatured at 94°C for 5 min and then annealed at 58°C for 4 min to allow the two fragments to complement each other and combine into a double strand, thus synthesizing the target fragment.

[0021] (3) The enzyme-digested vector fADL-le obtained in step (1) is ligated with the target fragment synthesized in step (2), and the reaction is carried out overnight at 16°C to obtain the recombinant plasmid;

[0022] (4) Transform the recombinant plasmid into Escherichia coli JM109 competent cells, select positive clones, and perform PCR identification.

[0023] (5) Use correctly identified positive clones to prepare phages displaying recombinant protein G3P1-12.

[0024] Furthermore, the recombinant protein G3P1-12 is used in the preparation of biological products for detecting serum p53 antibodies in tumor patients.

[0025] As can be seen from the above technical solution, compared with the prior art, this invention discloses a recombinant protein G3P1-12, its preparation method, and its application. Using phage display technology, a gene fragment encoding the N-terminal 1-12 peptide segment of the P53 protein is cloned into the PIII protein gene of a filamentous phage vector, preparing a recombinant protein displaying the P53 protein epitope. This recombinant protein is then displayed on the surface of a phage and used for the detection of p53 antibodies in the serum of cancer patients. This invention also discloses the application of recombinant protein G3P1-12 in the preparation of biological products for detecting p53 antibodies in the serum of cancer patients. These biological products have advantages such as high specificity, high sensitivity, simple preparation, and low cost. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 The attached figure shows the BglI restriction enzyme digestion results of the fADL-le vector of the present invention;

[0028] Wherein, 1 is the Marker; 2 is the fADL-le vector; and 3-5 are the fADL-le vectors digested with BglI.

[0029] Figure 2 The attached figure shows a positive clone of the recombinant fADL-le vector verified by PCR in this invention;

[0030] Wherein, 1 represents the marker; 2-5 represent positive clones;

[0031] Figure 3 The attached figure shows a partial sequencing peak diagram of the recombinant phage vector of the present invention;

[0032] Among them, nucleotides 881-916 are the nucleotide sequences encoding the target polypeptide;

[0033] Figure 4The attached figure shows the Western blot analysis of recombinant protein G3P1-12 according to this invention;

[0034] In this context, 1, 3, and 5 are markers; 2 is the hybridization of recombinant protein G3P1-12 with monoclonal antibodies against anti-PIII protein (positive control); and 4 and 6 are the hybridizations of recombinant protein G3P1-12 with serum from p53 antibody-positive tumor patients and healthy individuals, respectively (negative controls).

[0035] Figure 5 The attached figure shows the results of detecting serum P53 antibodies in breast cancer patients using the recombinant protein G3P1-12 of this invention. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1 Construction of recombinant bacteriophage

[0038] (1) Extraction of phage vector fADL-le

[0039] The commercially available phage vector fADL-le (purchased from Antibody Design laboratories, Catalog number: PD020) was extracted using Axygen's plasmid mini-extraction kit. The specific steps are as follows:

[0040] 1) Take 6 ml of bacterial culture JM109 that has been cultured overnight in LB medium (preserved in our laboratory, containing the vector fADL-le), centrifuge at 12000×g for 1 min, and discard the supernatant;

[0041] 2) Add 250 μl of Buffer S1 to suspend the bacterial precipitate. The suspension should be uniform and there should be no small bacterial clumps.

[0042] 3) Add 250 μl of Buffer S2, gently and thoroughly invert the container 4-6 times to mix evenly and allow the cells to fully lyse until a clear solution is formed;

[0043] 4) Add 350 μl of Buffer S3, gently and thoroughly invert 6-8 times, and centrifuge at 12000×g for 10 min;

[0044] 5) Take the supernatant from step 4) after centrifugation and transfer it to the preparation tube. Centrifuge at 12000×g for 1 min and discard the filtrate.

[0045] 6) Place the preparation tube back into the centrifuge tube, add 500 μl of Buffer W1, centrifuge at 12000×g for 1 min, and discard the filtrate;

[0046] 7) Place the preparation tube back into the centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12000×g for 1 min, and discard the filtrate; wash again with 700 μl of Buffer W2 in the same way, and discard the filtrate.

[0047] 8) Place the preparation tube back into the 2ml centrifuge tube and centrifuge at 12000×g for 1min;

[0048] 9) Transfer the preparation tube to a new 1.5 ml centrifuge tube, add 50 μl of Eluent solution to the center of the membrane in the preparation tube, let stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min.

[0049] (2) Enzyme digestion of fADL-le

[0050] The BglI enzyme digestion system for vector fADL-le is as follows:

[0051]

[0052] (3) Recovery of enzyme digestion vector

[0053] Agarose gel electrophoresis was performed on the vector fADL-le and the enzyme-digested vector fADL-le. The results are shown in the figure. Figure 1 The fADL-le vector mainly exists in circular and linear forms. After enzyme digestion, both become linear (only about 10 bp is cleaved). The electrophoresis rate is faster than that of the linear empty vector conformation, which initially proves that the enzyme digestion was successful.

[0054] Vectors that have been proven to be completely digested by agarose gel electrophoresis are then gel-cleaved and recovered, following the instructions of the DNA gel recovery kit:

[0055] 1) Cut off the agarose gel containing the target DNA under UV light, blot the liquid off the surface of the gel with a paper towel and cut it into pieces, calculate the weight of the gel, and use this weight as a gel volume;

[0056] 2) Add 3 gel volumes of Buffer DE-A, mix well, and heat at 75°C, mixing intermittently, until the gel is completely melted;

[0057] 3) Add 0.5 times the volume of Buffer DE-A to Buffer DE-B and mix thoroughly;

[0058] 4) Take the mixture from step 3) and transfer it to a DNA preparation tube. Centrifuge at 12000×g for 1 min and discard the filtrate.

[0059] 5) Place the preparation tube back into the 2ml centrifuge tube, add 500μl Buffer W1, centrifuge at 12000×g for 1min, and discard the filtrate;

[0060] 6) Place the preparation tube back into the centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12000×g for 1 min, and discard the filtrate; wash again with 700 μl of Buffer W2 in the same way, and discard the filtrate.

[0061] 7) Place the preparation tube back into the 2ml centrifuge tube and centrifuge at 12000×g for 1min;

[0062] 8) Transfer the preparation tube to a new 1.5 ml centrifuge tube, add 10 μl of Eluent solution to the center of the membrane in the preparation tube, let stand at room temperature for 1 min, and centrifuge at 12000×g for 1 min.

[0063] (4) Synthesis of phage display epitopes

[0064] Two complementary DNA fragments encoding the N-terminal amino acids MEEPQSDPSVEP of the P53 protein were synthesized.

[0065] 5'-CGGCCATGGCAATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCGGCCCGGG-3'; SEQ ID NO.3;

[0066] 5'-GGCCGGGCTCGACGCTAGGATCTGACTGCGGCTCCCTCCATTGCCATGGCCGGCT-3'; SEQ ID NO.4;

[0067] The two fragments were dissolved and mixed in equimolar amounts. The mixture was denatured at 94°C for 5 min and then annealed at 58°C for 4 min, so that the two fragments complement each other and combine to form a double strand.

[0068] (5) Ligation of the target fragment with the fADL-le restriction vector

[0069] The target fragment synthesized in step (4) was ligated to the fADL-le digested vector and reacted overnight at 16°C. The ligation reaction system is as follows:

[0070]

[0071] (6) Transformation of recombinant plasmids into E. coli JM109 competent cells

[0072] 1) Add 10 μl of ligation product to Escherichia coli JM109 competent cells, mix well, and incubate on ice for 30 min;

[0073] 2) 42℃, heat shock for 90 seconds;

[0074] 3) Immediately place on ice and in an ice bath for 10 minutes;

[0075] 4) Add 800 μl of LB medium, incubate at 37°C, 100 rpm for 45 min;

[0076] 5) Spread the transformation product evenly on LB solid medium containing kanamycin. After complete absorption, invert the medium and incubate overnight at 37°C for 12 hours.

[0077] 6) Select positive clones and perform PCR identification.

[0078] (7) Identification by bacterial culture PCR

[0079] Select single colonies and transfer them into 5 ml of LB liquid medium containing kanamycin resistance. Incubate at 37°C and 200 rpm for 8 h. Use 1 μl of the bacterial culture as a template for bacterial PCR identification. Primers and amplification conditions are as follows:

[0080] Upstream primer (PF): 5'-CCGTGCATCTGTCCTCGTTCAA-3'; SEQ ID NO.5;

[0081] Downstream primer (PR): 5'-GGGCTCGACGCTAGGATCTGACTG-3'; SEQ ID NO. 6;

[0082] An upstream primer PF was designed approximately 700 bp upstream of the pIII gene insertion site in the vector, and the antisense strand of the inserted fragment was used as a downstream primer for PCR verification.

[0083] The PCR reaction system is as follows:

[0084]

[0085] The PCR reaction conditions were: 94℃ pre-denaturation for 8 min; 35 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 30 s; and 72℃ extension for 10 min.

[0086] The amplification products were subjected to agarose gel electrophoresis, and the PCR verification results are shown in [link to PCR verification results]. Figure 2 Positive clones will show a specific target band at 700bp after PCR amplification, while negative clones will not show a specific band after PCR. Figure 2The results showed a specific target band at 700 bp, which preliminarily indicates that the coding fragment of the exogenous peptide (MEEPQSDPSVEP) was successfully inserted into the pIII gene of the phage, and the recombinant phage vector was successfully constructed.

[0087] After the reaction was completed, the selected positive clones were sent to Shanghai Sangon Biotech Service Co., Ltd. for sequencing.

[0088] The sequencing results of the recombinant vector are as follows: target fragment (the part highlighted in bold black and black) and Figure 3 The recombinant vector was successfully cloned into the PIII gene of the phage vector fADL-le, and it was completely identical to the original sequence being ligated, indicating that the recombinant vector was successfully constructed and can be used for the next step of preparing the recombinant protein G3P1-12.

[0089]

[0090]

[0091] Example 2 demonstrates the preparation of phages of recombinant protein G3P1-12.

[0092] 1) Inoculate 200 μl of correctly sequenced JM109 cells into 100 ml of LB liquid medium (100 μg / ml Karl Fibre). + The test tube was subjected to vigorous shaking at 37°C for 10 hours.

[0093] 2) 8000 rpm, 10 min, 4℃, retain the supernatant;

[0094] 4) Add one-sixth of the volume of PEG / NaCl solution, vortex, and incubate overnight at 4°C;

[0095] 5) Centrifuge at 12000 rpm for 15 min, and dissolve the phage pellet in 1 ml of TBS;

[0096] 6) Transfer the solution to a 1.5 ml EP tube, centrifuge at 14000 rpm for 1 min, carefully transfer the supernatant to a 1.5 ml EP tube, add 150 μl of PEG / NaCl to each EP tube, mix well and incubate at 4 °C overnight;

[0097] 7) Centrifuge at 14000 rpm for 15 min, dissolve the phage precipitate with 100 μl TBS, and store at 4℃.

[0098] Example 3: Western blot analysis of recombinant protein G3P1-12 phage

[0099] To verify the successful fusion expression of the N-terminal 1-12 peptide of the P53 protein with PIII and its display on the surface of bacteriophages, the prepared bacteriophages were hybridized with anti-PIII monoclonal antibodies and serum from P53 antibody-positive tumor patients, respectively. The specific steps are as follows:

[0100] 1) After SDS-PAGE, cut off the separating gel and place it in transfer buffer;

[0101] 2) Cut the PVDF membrane and filter paper to the size of the adhesive. Soak the filter paper in the transfer buffer solution. Soak the PVDF membrane in methanol for 1 minute and then place it in the transfer buffer solution.

[0102] 3) 80V constant voltage electric rotation for 2 hours;

[0103] 4) After staining with Ponceau S, cut the membrane into small strips to separate the proteins on the membrane from each other;

[0104] 5) Immerse the membrane in the blocking solution for 1 hour;

[0105] 6) Wash three times with PBST, with a 5-minute interval between each wash;

[0106] 7) Add commercially available mouse anti-PIII monoclonal antibody (NEB) diluted 1:1000 or serum from p53 antibody-positive cancer patients diluted 1:100 or serum from healthy individuals diluted 1:100, and incubate at 37°C for 1 hour.

[0107] 8) Wash three times with PBST, with a 5-minute interval between each wash;

[0108] 9) After diluting goat anti-mouse IgG or goat anti-human IgG secondary antibody to the working concentration, place the membrane strip in it and incubate at 37°C for 45 minutes;

[0109] 10) Wash three times with PBST, with a 5-minute interval between each wash;

[0110] 11) After diluting the DAB colorimetric solution to the working concentration, place the membrane in the solution and develop the color for 10 minutes.

[0111] The results are as follows Figure 4 As shown, the recombinant protein G3P1-12 can specifically react with both anti-PIII monoclonal antibody and P53 antibody in the serum of tumor patients, and the target bands are at the same position. However, the recombinant protein does not cross-react with the serum of healthy individuals, indicating that the recombinant protein G3P1-12 is successfully expressed and can specifically recognize P53 antibody in the serum of tumor patients.

[0112] Example 4: ELISA results of G3P1-12 phage detection of serum P53 antibody in breast cancer patients.

[0113] (1) G3P1-12-ELISA

[0114] Using the recombinant protein G3P1-12 bacteriophage as the detection antigen, serum P53 antibodies were detected in 60 breast cancer patients and 60 healthy individuals (negative controls) using the ELISA method. The specific steps are as follows:

[0115] 1) A 96-well microplate was coated with a phage displaying recombinant protein G3P1-12 as the coating antigen (referred to as G3P1-12-ELISA) at a concentration of 30 μg / ml, 50 μl per well, and incubated overnight at 4°C in a humidified chamber.

[0116] 2) On the second day, wash three times with PBST buffer for 3 minutes each time, and then wash twice with PBS solution for 3 minutes each time;

[0117] 3) Add 200 μl of blocking solution to each well and seal at 37°C for 1 h;

[0118] 4) After washing, add 50 μl of serum from breast cancer patients or healthy individuals diluted at a ratio of 1:200 to each well, and react at 37°C for 1 h.

[0119] 5) After repeated washing, add 50 μl of HRP-labeled goat anti-human IgG secondary antibody diluted 1:5000 to each well and incubate at 37°C for 45 min;

[0120] 6) After repeated washing, add 100 μl of substrate colorimetric solution TMB, react at room temperature in the dark for 12 min, and then add 50 μl of 2M sulfuric acid to each well to terminate the reaction.

[0121] 7) The absorbance at OD450nm was measured using an ELISA reader. Each sample was tested in duplicate, and the average value of the results was taken.

[0122] (2) P53-ELISA

[0123] The method for detecting serum 53 antibodies using recombinant P53 protein (purchased from Abcam, Catalog number: ab82201) as the coating antigen is abbreviated as P53-ELISA. Except that the antigen coated on the ELISA plate is recombinant P53 protein at a concentration of 5 μg / μl, the entire experimental procedure is exactly the same as G3P1-12-ELISA.

[0124] (3) Establishment of cut-off value

[0125] Based on the established G3P1-12-ELISA and P53-ELISA detection systems, serum samples from 60 healthy individuals were tested, and the cut-off value for each detection method was established using the mean value + 2SD method.

[0126] The results are as follows Figure 5 As shown, among 60 breast cancer patients, phage displaying recombinant protein G3P1-12 detected P53 antibody-positive serum in 12 patients, with a detection rate of 20.00%, which is basically consistent with the detection efficiency of recombinant P53 protein (21.67%). The specificities of the two detection methods were 96.67% (G3P1-12-ELISA) and 95.00% (P53-ELISA), respectively. These results indicate that recombinant protein G3P1-12 has advantages such as high specificity, high sensitivity, and simple preparation in detecting P53 antibodies in the serum of breast cancer patients, and can be used for research applications in the detection of P53 antibodies in the serum of breast cancer patients.

[0127] Phage display technology links genotype and phenotype, enabling researchers to achieve in vitro control of protein conformation at the gene level and obtain expression products with good biological activity in vitro. This invention utilizes phage display technology to obtain a recombinant protein G3P1-12 that displays the p53 protein epitope (N segment 1-12 peptide) on the surface of a phage. The method of this invention has the advantages of simple preparation, low cost, and high sensitivity, and can be applied to the detection of p53 antibodies in the serum of cancer patients.

[0128] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. sequence list <110> Xinxiang University <120> A recombinant protein G3P1-12, its preparation method and application <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 448 <212> PRT <213> Artificial Sequence <400> 1 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Met Glu Glu Pro Gln Ser Asp Pro Ser Val 20 25 30 Glu Pro Gly Pro Gly Gly Leu Ser Leu Glu Ala Glu Thr Val Glu Ser 35 40 45 Cys Leu Ala Lys Pro His Thr Glu Asn Ser Phe Thr Asn Val Trp Lys 50 55 60 Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly Cys Leu Trp 65 70 75 80 Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp Glu Thr Gln Cys Tyr 85 90 95 Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro Glu Asn Glu Gly Gly 100 105 110 Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 115 120 125 Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile Pro Gly Tyr Thr Tyr 130 135 140 Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly Thr Glu Gln Asn Pro 145 150 155 160 Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln Pro Leu Asn Thr Phe 165 170 175 Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln Gly Ala Leu Thr Val 180 185 190 Tyr Thr Gly Thr Val Thr Gln Gly Thr Asp Pro Val Lys Thr Tyr Tyr 195 200 205 Gln Tyr Thr Pro Val Ser Ser Lys Ala Met Tyr Asp Ala Tyr Trp Asn 210 215 220 Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly Phe Asn Glu Asp Pro 225 230 235 240 Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro 245 250 255 Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu 260 265 270 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 275 280 285 Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys 290 295 300 Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn 305 310 315 320 Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp 325 330 335 Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala 340 345 350 Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met 355 360 365 Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg 370 375 380 Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Tyr Val 385 390 395 400 Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile 405 410 415 Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe 420 425 430 Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser 435 440 445 <210> 2 <211> 1344 <212> DNA <213> Artificial Sequence <400> 2 atgaaatacc tattgcctac ggcggccgct ggattgttat tactcgcggc ccagccggcc 60 atggcaatgg aggagccgca gtcagatcct agcgtcgagc ccggcccgggg aggctgtct 120 ctagaagccg aaactgttga aagttgttta gcaaaacctc atacagaaaa ttcatttact 180 aacgtctgga aagacgacaa aactttagat cgttacgcta actatgaggg ctgtctgtgg 240 aatgctacag gcgttgtggt ttgtactggt gacgaaactc agtgttacgg tacatgggtt 300 cctattgggc ttgctatccc tgaaaatgag ggtggtggct ctgagggtgg cggttctgag 360 ggtggcggtt ctgagggtgg cggtactaaa cctcctgagt acggtgatac acctattccg 420 ggctatactt atatcaaccc tctcgacggc acttatccgc ctggtactga gcaaaacccc 480 gctaatccta atccttctct tgaggagtct cagcctctta atactttcat gtttcagaat 540 aataggttcc gaaataggca gggtgcatta actgtttata cgggcactgt tactcaaggc 600 actgaccccg ttaaaactta ttaccagtac actcctgtat catcaaaagc catgtatgac 660 gcttactgga acggtaaatt cagagactgc gctttccatt ctggctttaa tgaggatcca 720 ttcgtttgtg aatatcaagg ccaatcgtct gacctgcctc aacctcctgt caatgctggc 780 ggcggctctg gtggtggttc tggtggcggc tctgagggtg gcggctctga gggtggcggt 840 tctgagggtg gcggctctga gggtggcggt tccggtggcg gctccggttc cggtgatttt 900 gattatgaaa aaatggcaaa cgctaataag ggggctatga ccgaaaatgc cgatgaaaac 960 gcgctacagt ctgacgctaa aggcaaactt gattctgtcg ctactgatta cggtgctgct 1020 atcgatggtt tcattggtga cgtttccggc cttgctaatg gtaatggtgc tactggtgat 1080 tttgctggct ctaattccca aatggctcaa gtcggtgacg gtgataattc acctttaatg 1140 aataatttcc gtcaatattt accttctttg cctcagtcgg ttgaatgtcg cccttatgtc 1200 tttggcgctg gtaaaccata tgaattttct attgattgtg acaaaataaa cttattccgt 1260 ggtgtctttg cgtttctttt atatgttgcc acctttatgt atgtattttc gacgtttgct 1320 aacatactgc gtaataagga gtct 1344 <210> 3 <211> 55 <212> DNA <213> Artificial Sequence <400> 3 cggccatggc aatggaggag ccgcagtcag atcctagcgt cgagcccggc ccggg 55 <210> 4 <211> 55 <212> DNA <213> Artificial Sequence <400> 4 gggccgggct cgacgctagg atctgactgc ggctcctcca ttgccatggc cggct 55 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 ccgtgcatct gtcctcgttc aa 22 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <400> 6 gggctcgacg ctaggatctg actg 24 <210> 7 <211> 966 <212> DNA <213> Artificial Sequence <400> 7 tcctgactgg tataatgagc cagttcttaa aatcgcataa ggtaattcaa aatgattaaa 60 gttgaaatta aaccatctca agcgcaattt actacccgtt ctggtgtttc tcgtcagggc 120 aagccttatt cactgaatga gcagctttgt tacgttgatt tgggtaatga atatccggtg 180 cttgtcaaga ttactctcga cgaaggtcag ccagcgtatg cgcctggtct gtacaccgtg 240 catctgtcct cgttcaaagt tggtcagttc ggttctctta tgattgaccg tctgcgcctc 300 gttccggcta agtaacatgg agcaggtcgc ggatttcgac acaatttatc aggcgatgat 360 acaaatctcc gttgtacttt gtttcgcgct tggtataatc gctgggggtc aaagatgagt 420 gttttagtgt attctttcgc ctctttcgtt ttaggttggt gccttcgtag tggcattacg 480 tattttaccc gtttaatgga aacttcctca tgaaaaagtc tttagtcctc aaagcctccg 540 tagccgttgc taccctcgtt ccgatgctgt ctttcgctgc tgagggtgac gatcccgcaa 600 aagcggcctt taactccctg caagcctcag cgaccgaata tatcggttat gcgtgggcga 660 tggttgttgt cattgtcggc gcaactatcg gtatcaagct gtttaagaaa ttcacctcga 720 aagcaagctg ataaaccgat acaattaaag gctccttttg gagccttttt tttgtcgact 780 aacgagggca aatcatgaaa tacctattgc ctacggcggc cgctggattg ttattactcg 840 cggcccagcc ggccatggca atggaggagc cgcagtcaga tcctagcgtc gagcccggcc 900 cgggaggcct gtctctagaa gccgaaactg ttgaaagttg tttagcaaaa cctcatacag 960 aaaatc 966

Claims

1. A recombinant protein G3P1-12, characterized in that, The amino acid sequence MEEPQSDPSVEP from position 1 to 12 of the N segment of the P53 protein is displayed on the minor coat protein PIII of the filamentous phage; the amino acid sequence of the recombinant protein G3P1-12 is shown in SEQ ID NO.1; The specific steps for preparing the recombinant protein G3P1-12 are as follows: (1) The vector fADL-le was digested with Bgl I enzyme and the digested vector fADL-le was recovered; (2) Synthesize the target nucleic acid fragment encoding the phage display epitope MEEPQSDPSVEP: Two complementary DNA fragments encoding the N-terminal amino acids MEEPQSDPSVEP of the P53 protein were synthesized. 5'-CGGCCATGGCAATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCGGCCCGGG-3'; SEQ ID NO.3; 5'-GGCCGGGCTCGACGCTAGGATCTGACTGCGGCTCCCTCCATTGCCATGGCCGGCT-3'; SEQ ID NO.4; The two fragments were dissolved and mixed in equimolar amounts. The mixture was denatured at 94°C for 5 min and then annealed at 58°C for 4 min to allow the two fragments to complement each other and combine into a double strand, thus synthesizing the target fragment. (3) The enzyme-digested vector fADL-le obtained in step (1) is ligated with the target fragment synthesized in step (2), and the reaction is carried out overnight at 16°C to obtain the recombinant plasmid; (4) Transform the recombinant plasmid into Escherichia coli JM109 competent cells, select positive clones, and perform PCR identification. (5) Use correctly identified positive clones to prepare phages displaying recombinant protein G3P1-12.

2. The recombinant protein G 3P1-12 according to claim 1, characterized in that, The nucleotide sequence encoding the recombinant protein G3P1-12 is shown in SEQ ID NO.

2.

3. The application of the recombinant protein G3P1-12 according to claims 1-2 in the preparation of phages for detecting p53 antibodies in the serum of tumor patients.

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