LU501601B1 - CODING SEQUENCE OF RECOMBINANT PROTEIN, RECOMBINANT PROTEIN, AND PREPARATION METHOD OF MONOCLONAL ANTIBODY (mAb) FOR RECOMBINANT PROTEIN - Google Patents

Abstract

The present disclosure discloses a coding sequence of a recombinant protein, a recombinant protein, and a preparation method of a monoclonal antibody (mAb) for the recombinant protein. The recombinant protein includes four tag epitopes: 6xHis, S Tag, Trx, and GST. In combination with a preferred codon of Escherichia coli (E. coli), the recombinant amino acid sequence is converted into a corresponding nucleotide sequence. A recombinant antigen is prepared by molecular biology technology and used to immunize mice, and high-quality monoclonal cell lines for the four tag proteins are prepared through a screening platform such as enzyme-linked immunosorbent assay (ELISA).

Description

BL-5338

CODING SEQUENCE OF RECOMBINANT PROTEIN, RECOMBINANT PROTEIN, ~ 60

AND PREPARATION METHOD OF MONOCLONAL ANTIBODY (mAb) FOR

RECOMBINANT PROTEIN

TECHNICAL FIELD

[01] The present disclosure belongs to the field of biotechnology, and specifically relates to a coding sequence of a recombinant protein, a recombinant protein, and a preparation method of a monoclonal antibody (mAb) for the recombinant protein.

BACKGROUND ART

[02] Tag-antibodies are a group of affinity-purified mouse mAbs. The tag-antibodies can be used to detect tag sequences (such as MyC, His, GST, and HA) on various commercial expression vectors to analyze expression levels and functions of target proteins. With the principle of antigen-antibody reaction, these tag-antibodies can bind to corresponding tag fusion proteins with high specificity. Tag-antibodies are common tools for the study on protein expression, signal transduction, and gene function.

[03] An immunogen used in the preparation of conventional tag-mAbs is an intact protein expressed through genetic engineering. However, due to base codons, the protein is difficult to express in Escherichia coli (E. coli), with an extremely-low expression level, which makes the subsequent purification work difficult and seriously hinders the preparation of corresponding mAbs. In addition, there are many types of tag proteins, and each tag protein needs to be prepared separately, with high time consumption and high cost, which is not conducive to the widespread promotion and use on the market.

SUMMARY

[04] In order to avoid the deficiencies in the background art, the present disclosure designs a recombinant protein and a mAb therefor. In the present disclosure, a variety of tag-mAbs can be prepared simultaneously, which enhances the detection sensitivity.

[05] In order to achieve the above objective, the present disclosure provides the following technical solutions:

[06] A coding sequence of the recombinant protein is shown in SEQ ID NO. 1, specifically as follows:

[07] atgagcccga ttetgggcta ttggaaaatt aaaggectgg tgcagecgac cegectgetg

[08] ctggaatatc tggaagaaaa atatgaagaa catctgtatg aacgcgatga aggcgataaa

[09] tggcgcaaca aaaaatttga actgggcctg gaatttccga acctgecgta ttatattgat 1

BL-5338

[10] ggcgatgtga aactgaccca gageatggec attattegct atattgecga taaacataac 750760)

[11] atgctgggceg getgeccgaa agaacgcgcc gaaattagca tgetggaagg Cgccgtgctg

[12] —gatattcget atggegtgag cegcattgce tatagcaaag attttgaaac cctgaaagtg

[13] gattttctga gcaaactgee ggaaatectg aaaatgtttg aagatcgect gtgccataaa

[14] acctatctga acggcgatca tgtgacccat ccggatttta tgetgtatga tgecctggat

[15] gtggtectet atatggatec gatgtgectg gatgectttc cgaaactggt gtectttaaa

[16] aaacgcattg aagccattec gcagattgat aaatatctga aaagcagcaa atatattgec

[17] — tggccgctgc agggctggca ggccaccttt ggcggcggcg atcatecgec gaaaagegat

[18] ctggtgccgc gcggcagece gggcattcat cgegatggeg geggeggege cgtgcatetg

[19] — ctggcctttg aatttagcag cageggcttt tttggcggcg gcggcggcca ggtgagegtg

[20] — gaagaactgt ttcagctgac ctttcgccag tgcacccatt ttggetgeeg ceattttace

[21] gtgtttgaac agcagcagag ccgegatace accgatgcca tttttcgccg cagegeecge

[22] gtgctgattg atgtgcagtt ttgcaacgge cagtttgccec tgatttttgt gagegatttt

[23] attcagaacc gcggcgatca ttttgcccgc accgecccge tgtgeccgga aattgatgaa

[24] — gatcgcccgg tgegcetttga atatattcge gtgaaaaccg tggtgagcca ggtgaacaac

[25] tttattgccc atggeggegg cggccatcat catcatcate at.

[26] The recombinant protein has an amino acid sequence shown in SEQ ID NO. 2, specifically as follows:

[27] Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

[28] Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

[29] Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

[30] Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

[31] Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

[32] Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

[33] Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

[34] Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

[35] Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

[36] Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

[37] Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

[38] Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

[39] Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

[40] Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg

[41] Gly Ser Pro Gly Ile His Arg Asp Gly Gly Gly Gly Ala Val His Val

[42] Leu Ala Phe Glu Phe Ser Ser Ser Gly Phe Phe Gly Gly Gly Gly Gly

[43] Gln Val Ser Val Glu Glu Leu Phe Gln Leu Thr Phe Arg Gln Cys Thr 2

BL-5338

[44] His Phe Gly Cys Arg His Phe Thr Val Phe Glu Gln Gln Gln Ser Arg 750760)

[45] Asp Thr Thr Asp Ala Ile Phe Arg Arg Ser Ala Arg Val Leu Ile Asp

[46] Val Gln Phe Cys Asn Gly Gln Phe Ala Leu Ile Phe Val Ser Asp Phe

[47] Ile Gln Asn Arg Gly Asp His Phe Ala Arg Thr Ala Pro Leu Cys Pro

[48] Glu Ile Asp Glu Asp Arg Pro Val Arg Phe Glu Tyr Ile Arg Val Lys

[49] Thr Val Val Ser Gln Val Asn Asn Phe Ile Ala His Gly Gly Gly Gly

[50] His His His His His His.

[51] The recombinant protein expression vector is a plasmid vector carrying the coding sequence shown in SEQ ID NO. 1.

[52] The recombinant protein and the recombinant protein expression vector can be used in the preparation of a tag-mAb.

[53] The present disclosure also provides a preparation method of a tag-mAb, including the following steps:

[54] (1) synthesizing a recombinant protein coding sequence shown in SEQ ID NO. 1, and linking the recombinant protein coding sequence to a plasmid vector to construct a recombinant protein expression vector;

[55] (2) transforming the recombinant protein expression vector into Æ. coli, and screening to obtain a recombinant protein-expressing strain;

[56] (3) subjecting the recombinant protein-expressing strain to large-scale cultivation, and conducting purification to obtain a recombinant protein, where the recombinant protein has an amino acid sequence shown in SEQ ID NO. 2; and

[57] (4) immunizing Balb/c mice with the recombinant protein multiple times, fusing mouse spleen cells with sp2/0 myeloma cells, and conducting multiple rounds of screening and multi-index identification to obtain a monoclonal cell line for a tag protein corresponding to the recombinant protein; and preparing the tag-mAb by the monoclonal cell line for the tag protein.

[58] The present disclosure will be further described below:

[59] 1. The present disclosure uses 4 tag-binding proteins (6xHis, S Tag, Trx, and GST) as target antigens. Sequence alignment results show that the selected epitopes have no obvious homology with other protein sequences.

[60] 2. In order to promote the stimulation of the selected epitopes on the immune system of

BALB/c mice and enhance the immune effect, the selected four dominant epitope sequences are repeated and then linked through flexible fragments (four consecutive glycines) to form an amino acid sequence of the recombinant protein.

[61] 3. With a preferred codon of Æ. coli, the amino acid sequence of the recombinant protein is converted into the corresponding nucleotide sequence, which facilitates the expression of the 3

BL-5338 recombinant protein in Æ. coli to increase an expression level. 750760)

[62] 4. The nucleotide sequence obtained in the previous step is chemically synthesized and inserted into the expression vector PET-28a(+) through enzyme digestion and ligation to construct a recombinant protein expression vector.

[63] 5. The recombinant protein expression vector is transformed into Æ. coli ER2566 competent cells, and then the cells are screened to obtain a recombinant protein-expressing strain.

[64] 6. The recombinant protein-expressing strain is subjected to large-scale cultivation, ultrasonic disruption, and low-temperature centrifugation, a resulting supernatant is subjected to affinity chromatography on a nickel agarose affinity chromatographic column, and a purified recombinant protein is obtained through elution.

[65] 7. BALB/c mice are immunized with the purified recombinant protein multiple times, then spleen cells are collected and fused with sp2/0 myeloma cells, and multiple rounds of screening are conducted to finally obtain a hybridoma cell line.

[66] 8. BALB/c mouse ascites is prepared by the hybridoma cell line, and a mAb is purified in two steps using the caprylic acid-ammonium sulfate method and the Protein G method.

[67] Compared with the prior art, the present disclosure has the following beneficial effects.

[68] 1. The repeated and tandem expression of four tag-binding protein epitopes is achieved through the molecular biology technology, which enhances the stimulation of the target epitope on the mouse immune system and eliminates the interference that may be caused by unrelated sequences.

[69] 2. The recombinant protein as an immunogen only includes four tag protein epitopes, which ensures that an mAb finally obtained only specifically recognizes the four tag proteins; and screening is conducted to obtain the four tag proteins at the same time, which improves the detection sensitivity and reduces the experimental cost.

[70] 3. The preferred codon of E. coli is used to optimize the nucleotide sequence corresponding to the recombinant protein, thereby greatly improving an expression level of the recombinant protein in Æ. coli.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[71] 1. Selection of 4 tag protein epitopes

[72] 4 tag proteins were used as target antigens, the biological software DNAssist2.0 was used to analyze the hydrophilicity and antigenicity of epitope sequences of the tag proteins, and the 6xHis, S Tag, Trx, and GST epitopes A, B, C, and D were selected, respectively. Moreover, sequence alignment results showed that the selected epitopes A, B, C, and D had high sequence 4

BL-5338 specificity and showed no obvious homology with other protein sequences. HUS01601

[73] 2. Concatenation of the 4 tag protein epitopes

[74] In order to enhance the stimulation of the selected epitopes on the mouse immune system to facilitate subsequent experiments, the four tag protein epitope sequences A, B, C, and D were linked through flexible fragments (four consecutive glycines) to obtain an amino acid sequence of a recombinant protein, as shown in SEQ ID No. 2 in the sequence listing.

[75] 3. Optimization of a nucleotide sequence encoding the recombinant protein

[76] In order to increase an expression level of the recombinant protein in E. coli, on the premise that the amino acid sequence of the recombinant protein remained unchanged, the amino acid sequence of the recombinant protein was converted into the corresponding nucleotide sequence according to a preferred codon of Æ. coli. The nucleotide sequence was specifically shown in SEQ ID No: 1. After nucleotide sequences corresponded to restriction sites BamHI and

EcoRI were added upstream and downstream, the nucleotide sequence was synthesized by

Hangzhou Goodhere Biotechnology Co., Ltd. A synthesized target gene was cloned to a pMD19-T vector (Takara Biotechnology (Dalian) Co., Ltd.).

[77] 4. Construction of a recombinant protein expression vector

[78] The pMD19-T vector carrying the target gene and the PET-28a(+) vector (Novagen,

Germany) were subjected to double enzyme digestion at 37°C for 12 h with restriction enzymes

BamHI and EcoRI (Takara Biotechnology (Dalian) Co., Ltd.). Enzyme digestion products were subjected to 1% agarose gel electrophoresis, and the target gene and the PET-28a(+) vector were subjected to gel extraction (the gel extraction kits used in the present disclosure were from

Ningbo ZD Biotechnology Co., Ltd.). The recovered target gene and pET-28a(+) vector were subjected to ligation for 12 h at 4°C in a given ratio by T4 ligase (Takara Biotechnology (Dalian)

Co., Ltd.); a ligation product was transformed into DH5a competent cells (Hangzhou Goodhere

Biotechnology Co., Ltd.), and the cells were plated on a kanamycin (50 pg/mL) resistant LB plate and cultivated for 12 h at 37°C; a monoclonal strain was picked from the plate and added to a kanamycin (50 pg/mL) resistant LB liquid medium; and after the monoclonal strain was cultivated on a constant temperature shaker for 12 h at 37°C, plasmids were extracted by a plasmid purification kit (all plasmid purification kits used in the present disclosure were purchased from Ningbo ZD Biotechnology Co., Ltd), and double enzyme digestion identification was conducted with BamHI and EcoRI to obtain a correct recombinant expression vector.

[79] 5. Construction of a recombinant protein E-expressing strain

[80] The constructed recombinant expression vector was transformed into Æ coli ER2566 competent cells, and the cells were plated on a kanamycin (50 pg/mL) resistant LB plate and

BL-5338 cultivated overnight at 37°C. The next day, a monoclonal strain was picked from the plate, added 201601 to a kanamycin (50 pg/mL) resistant LB liquid medium, and cultivated on a constant temperature shaker for 8 h at 37°C; and isopropyl-B-D-thiogalactoside (IPTG) (final concentration: 1.0 mmol/L) was added to conduct induced expression for 4 h, and then a protein electrophoresis sample was prepared. Results of 13.5% polyacrylamide gel electrophoresis (PAGE) indicated that a recombinant protein was expressed successfully and a recombinant protein-expressing strain was obtained.

[81] ©. Purification of a recombinant protein F

[82] The recombinant protein-expressing strain was inoculated into an LB liquid medium, kanamycin was added at a final concentration of 50 ug/mL, and the strain was cultivated on a constant temperature shaker for 8 h at 37°C; a resulting bacterial solution was diluted in 1:100 with an LB liquid medium including 50 pg/mL kanamycin, dispensed into bacterial culture bottles, and cultivated on a constant temperature shaker at 37°C until an OD600 was 0.8; and

IPTG was added at a final concentration of 1.0 mmol/L, and induced cultivation was further conducted for 4 h. Bacteria were collected by centrifugation, and then subjected to low-temperature ultrasonic disruption and low-temperature centrifugation, a resulting supernatant was passed through a nickel agarose affinity chromatographic column, and a purified recombinant protein was finally obtained through washing and elution.

[83] 7. Construction of a coupling protein for detection

[84] A cysteine was linked to the N terminus of each of the two tag protein epitope sequences

A and B to synthesize polypeptide sequences G and H. BSA (Roche) was selected as a vector protein, and the SPDP (PIERCE) coupling method was used to couple each of the synthesized polypeptide sequences with BSA: 4.6 mg of SPDP was dissolved in 740 ul of DMSO, with a final concentration of 20 mM; 0.1008 g of BSA was dissolved in 2 ml of a PBS-EDTA solution, and a resulting solution stood at room temperature for 1 h; the HiTrapTM Deaslting column was used to elute excess SPDP; and 4 mg of a polypeptide was added to the coupled BSA-SPDP system, and a resulting mixture was kept overnight at room temperature to obtain products

BSA-G and BSA-H (which were synthesized by Hangzhou Goodhere Biotechnology Co., Ltd.).

The proteins GST and TRX (purchased from Hangzhou Beaconlab Biotechnology Co., Ltd.) were named I and L, respectively.

[85] 8. Acquisition of a hybridoma cell line

[86] 5 to 7-week-old female BALB/c mice were selected and each subcutaneously injected (multi-point injection) with 70 pg of the recombinant protein that was emulsified with Freund's complete adjuvant (FCA), 15 d later, booster immunization was conducted, that is, the same amount of the recombinant protein was emulsified with Freund's incomplete adjuvant (FICA) 6

BL-5338 and a resulting emulsion was subcutaneously injected through multi-point injection; and further 201601 d later, the third booster immunization was conducted by the same method as the second immunization. 30 d later, 120 ug of the recombinant protein was intraperitoneally injected for booster; and 72 h after the intraperitoneal injection for booster, blood was collected from the orbit and the mice were sacrificed; the spleen was collected to prepare a cell suspension, and cells were counted; Sp2/0 mouse myeloma cells in well growth were collected at an amount 1/5 of the spleen cells and then mixed with the spleen cells; a resulting mixture was centrifuged, and then polyethylene glycol (PEG-4000) was added for the fusion of the two; then an equal volume of feeder cells was added, and a resulting mixture was thoroughly mixed, added to a 96-well cell plate (200 uL/well), and cultivated in a 5% carbon dioxide incubator; and 5 d later, the medium was half-replaced, and 10 d later, a hybridoma cell culture supernatant in the 96-well cell culture plate was tested through indirect ELISA.

[87] The specific method was as follows:

[88] The proteins BSA-G, BSA-H, I, and L were each diluted with a coating buffer (with a final concentration of 1 pg/mL), and then added to a microtiter plate (Shenzhen Jincanhua

Industries Co., Ltd.) at 100 uL/well to coat at 4°C for 12 h; then the microtiter plate was washed once with a washing liquid and pat-dried; a blocking buffer was added at 150 uL/well to block at 37°C for 2 h, a liquid in the well was removed, and the microtiter plate was pat-dried; a cell culture supernatant and control serum were each added at 100 pL/well, and then the microtiter plate was incubated at 37°C for 1 h; the microtiter plate was washed three times with a washing liquid and then pat-dried; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was added at 100 uL/well, and then the microtiter plate was incubated at 37°C for 30 min, washed four times with a washing liquid, and pat-dried; 50 pL of each of a chromogenic solution A and a chromogenic solution B was added to each well, and then the microtiter plate was incubated at 37°C for 10 min in the dark; a stop solution was added at 50 uL/well to stop the chromogenic reaction; and after zeroing with the blank well, an OD value was read on a microplate reader at 450 nm. Serum of immunized mice was used as a positive control, and relevant solutions had the following formulas:

[89] coating buffer: Na,COs: 1.5 g, and NaHCOs: 2.9 g, which was diluted to 1,000 mL (pH 9.6) with double distilled water (DDW);

[90] blocking buffer: Na,HPO4 12H,0: 2.68 g, NaH,PO4-2H,0: 0.39 g, NaCl: 8.5 g, and bovine serum albumin (BSA): 20 g, which was diluted to 1,000 mL (pH 7.4) with DDW;

[91] washing liquid: Na,HPO4 12H,O: 2.68 g, NaH,PO4-2H,O: 0.39 g, NaCl: 8.5 g, and

Tween-20: 0.5 mL, which was diluted to 1,000 mL (pH 7.4) with DDW;

[92] chromogenic solution A: dissolving 200 mg of tetramethylbenzidine (TMB) in 100 mL 7

BL-5338 of absolute ethanol, and diluting a resulting solution to 1,000 mL with DDW; HUS0T601

[93] chromogenic solution B: citric acid: 2.1 g, and Na;HPO4 12H,O: 71 g, which was diluted to 1,000 mL with DDW;

[94] when in use: 1 mL of chromogenic solution A + 1 mL of chromogenic solution B + 0.4 uL of 30% H:0>; and

[95] stop solution: 2 M H,SO4, 21.7 mL of concentrated H:SO4, which was diluted to 1,000 mL with DDW.

[96] For positive hybridoma cell clones, the limiting dilution method was used for subcloning.

The subcloning was conducted three times, and screening was conducted to obtain 6xHis 3 hybridoma cell lines (1F4, 8B6, and 3D7), S Tag 2 hybridoma cell lines (SD6 and 2C3), Trx 5 hybridoma cell lines (4F4, 6B7, 3G7, 6T5, and 7H5), and GST3 hybridoma cell lines (7F4, 2C7, and 6D7).

[97] BALB/c mouse ascites was prepared by the hybridoma cell lines, and a mAb was purified in two steps using the caprylic acid-ammonium sulfate method and the Protein G method.

[98] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure. 8

SEQUENCE LISTING LU501601 <110> XIANGYA HOSPITAL CENTRAL SOUTH UNIVERSITY <120> CODING SEQUENCE OF RECOMBINANT PROTEIN, RECOMBINANT PROTEIN, AND

PREPARATION METHOD OF MONOCLONAL ANTIBODY (mAb) FOR RECOMBINANT

PROTEIN

<130> HKIPCTU202109975 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1122 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the recombinant protein <400> 1 atgagcccga ttctgggcta ttggaaaatt aaaggcctgg tgcagccgac ccgectgetg 60 ctggaatatc tggaagaaaa atatgaagaa catctgtatg aacgcgatga aggcgataaa 120 tggcgcaaca aaaaatttga actgggcctg gaatttccga acctgccgta ttatattgat 180 ggcgatgtga aactgaccca gagcatggcc attattcgct atattgccga taaacataac 240 atgctgggcg gctgcccgaa agaacgcgcc gaaattagca tgctggaagg cgecegtgetg 300 gatattcgct atggcgtgag ccgcattgcc tatagcaaag attttgaaac cctgaaagtg 360 gattttctga gcaaactgcc ggaaatgctg aaaatgtttg aagatcgcct gtgccataaa 420 acctatctga acggcgatca tgtgacccat ccggatttta tgctgtatga tgccctggat 480 gtggtgectgt atatggatcc gatgtgcctg gatgecctttc cgaaactggt gtgctttaaa 540 aaacgcattg aagccattcc gcagattgat aaatatctga aaagcagcaa atatattgcc 600 tggccgctgc agggctggca ggccaccttt ggcggcggcg atcatccgec gaaaagcgat 660 ctggtgccgc gcggcagcee gggcattcat cgecgatggeg geggeggege cgtgeatgtg 720 ctggcctttg aatttagcag cagcggcttt tttggcggcg gcggcggcca ggtgagcgtg 780 gaagaactgt ttcagctgac ctttcgccag tgcacccatt ttggctgccg ccattttacc 840 gtgtttgaac agcagcagag ccgcgatacc accgatgcca tttttcgccg cagcgcccgc 900 gtgctgattg atgtgcagtt ttgcaacggc cagtttgccc tgatttttgt gagcgatttt 960 attcagaacc gcggcgatca ttttgeecege accgececcgc tgtgcccgga aattgatgaa 1020 gatcgcccgg tgcgctttga atatattcgc gtgaaaaccg tggtgagcca ggtgaacaac 1080 tttattgccc atggcggcgg cggccatcat catcatcatc at 1122 <210> 2 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the recombinant protein <400> 2

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu LU501601 165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg 210 215 220

Gly Ser Pro Gly Ile His Arg Asp Gly Gly Gly Gly Ala Val His Val 225 230 235 240

Leu Ala Phe Glu Phe Ser Ser Ser Gly Phe Phe Gly Gly Gly Gly Gly 245 250 255

Gln Val Ser Val Glu Glu Leu Phe Gln Leu Thr Phe Arg Gln Cys Thr 260 265 270

His Phe Gly Cys Arg His Phe Thr Val Phe Glu Gln Gln Gln Ser Arg 275 280 285

Asp Thr Thr Asp Ala Ile Phe Arg Arg Ser Ala Arg Val Leu Ile Asp 290 295 300

Val Gln Phe Cys Asn Gly Gln Phe Ala Leu Ile Phe Val Ser Asp Phe 305 310 315 320

Ile Gln Asn Arg Gly Asp His Phe Ala Arg Thr Ala Pro Leu Cys Pro 325 330 335

Glu Ile Asp Glu Asp Arg Pro Val Arg Phe Glu Tyr Ile Arg Val Lys 340 345 350

Thr Val Val Ser Gln Val Asn Asn Phe Ile Ala His Gly Gly Gly Gly 355 360 365

His His His His His His 370

Claims (7)

BL-5338 LU501601 WHAT IS CLAIMED IS:

1. A coding sequence of a recombinant protein, wherein the coding sequence is shown in SEQ ID NO. 1.

2. A recombinant protein encoded by the coding sequence according to claim 1, wherein the recombinant protein has an amino acid sequence shown in SEQ ID NO. 2.

3. A recombinant protein expression vector, wherein the recombinant protein expression vector is a plasmid vector carrying the coding sequence shown in SEQ ID NO. 1.

4. The recombinant protein expression vector according to claim 3, wherein an original plasmid for the recombinant protein expression vector is PET-28a(+).

5. Use of the recombinant protein according to claim 2 in the preparation of a tag-monoclonal antibody (mAb).

6. Use of the recombinant protein expression vector according to claim 3 or 4 in the preparation of a tag-mAb.

7. A preparation method of a tag-mAb, comprising the following steps: (1) synthesizing a recombinant protein coding sequence shown in SEQ ID NO. 1, and linking the recombinant protein coding sequence to a plasmid vector to construct a recombinant protein expression vector; (2) transforming the recombinant protein expression vector into Escherichia coli (E. coli), and screening to obtain a recombinant protein-expressing strain; (3) subjecting the recombinant protein-expressing strain to large-scale cultivation, and conducting purification to obtain a recombinant protein, wherein the recombinant protein has an amino acid sequence shown in SEQ ID NO. 2; and (4) immunizing Balb/c mice with the recombinant protein multiple times, fusing mouse spleen cells with sp2/0 myeloma cells, and conducting multiple rounds of screening and multi-index identification to obtain a monoclonal cell line for a tag protein corresponding to the recombinant protein; and preparing the tag-mAb by the monoclonal cell line for the tag protein. 9