Pyrrolysine aminoacyl-tRNA synthetase mutants and their uses

Abstract

The present invention provides a pyrrolysyl - tRNA synthetase mutant and its use. The pyrrolysyl - tRNA synthetase mutant provided by the present invention has a more efficient recognition and catalytic efficiency for unnatural amino acids, thereby increasing the expression efficiency of recombinant proteins by 230% - 380%, which is beneficial to reducing the production cost of recombinant proteins and realizing the large - scale commercial production of recombinant proteins.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a pyrrolysine aminoacyl-tRNA synthetase mutant, a corresponding expression vector and use thereof, and a method for preparing a recombinant protein containing a non-natural amino acid. Background Art

[0002] The genetic code expansion technology (GCE) has achieved remarkable achievements in the past two decades and has been increasingly widely used in the field of biotechnology. The basic applications of GCE include protein structure and function analysis, discovery of interacting ligands, simulation of post-translational modifications, enzyme activity regulation, protein function regulation, and live cell imaging; its applications in drug research include the development of antibody-drug conjugates (ADCs), bispecific antibodies (BsAbs), chimeric antigen receptor T cells (CAR-T), vaccines, and long-acting protein drugs. Among them, ADC drugs applying GCE technology have more uniform and stable physicochemical properties, and ADC projects targeting HER2, PSMA, CD70, FolRα and other targets have successively entered the clinical research stage. Thus, it can be seen that introducing non-natural amino acids into protein drugs using GCE technology has very important and broad application potential.

[0003] A non-natural amino acid NPAK is disclosed in Chinese Patent (CN113582881B), and its structural formula is shown as formula (I):

[0004]

[0005] In this patent, Methanosarcina barkeri pyrrolysine aminoacyl-tRNA synthetase (abbreviated as MbPylRS) was used to achieve site-directed introduction of NPAK into recombinant human growth hormone. However, in practical applications, there are still disadvantages such as limited activity of MbPylRS in recognizing NPAK and low yield of target protein, resulting in high costs and difficult product quality control in commercial large-scale production.

[0006] Therefore, there is a need to develop an aminoacyl-tRNA synthetase mutant that can efficiently recognize NPAK. Summary of the Invention

[0007] The purpose of the present invention is to provide a pyrrolysine aminoacyl-tRNA synthetase mutant that can efficiently recognize NPAK, its coding nucleic acid molecule, a corresponding expression vector and use thereof, aiming to solve the problem of low activity of natural aminoacyl-tRNA synthetase in recognizing NPAK existing in the prior art.

[0008] The pyrrolysyl - tRNA synthetase mutant provided by the present invention is a mutant of pyrrolysyl - tRNA synthetase from Methanomethylophilus alvus (hereinafter sometimes abbreviated as MaPylRS mutant or mutant). The mutant is obtained by mutating the amino acid residues in the wild - type pyrrolysyl - tRNA synthetase (MaPylRS, whose amino acid sequence is shown in SEQ ID NO:1 and Sequence ID in NCBI is WP_015505008.1) of Methanomethylophilus alvus.

[0009] Specifically, in a first aspect, the present invention provides a pyrrolysyl - tRNA synthetase mutant, which is an enzyme mutant obtained by performing the following amino acid substitutions on the amino acid residues at position 126 and / or position 129 in the amino acid sequence of pyrrolysyl - tRNA synthetase shown in SEQ ID NO:1. The amino acid substitutions are as follows:

[0010] The tyrosine at position 126 is replaced with alanine;

[0011] The methionine at position 129 is replaced with leucine.

[0012] In some embodiments, the pyrrolysyl - tRNA synthetase mutant is a mutant obtained by performing an amino acid substitution, and the amino acid substitution includes replacing the tyrosine at position 126 in the amino acid sequence of pyrrolysyl - tRNA synthetase shown in SEQ ID NO:1 with alanine.

[0013] In some embodiments, the pyrrolysyl - tRNA synthetase mutant is a mutant obtained by performing an amino acid substitution, and the amino acid substitution includes replacing the methionine at position 129 in the amino acid sequence of pyrrolysyl - tRNA synthetase shown in SEQ ID NO:1 with leucine.

[0014] In some embodiments, the pyrrolysyl - tRNA synthetase mutant is a mutant obtained by performing an amino acid substitution, and the amino acid substitution includes replacing the tyrosine at position 126 in the amino acid sequence of pyrrolysyl - tRNA synthetase shown in SEQ ID NO:1 with alanine and replacing the methionine at position 129 with leucine.

[0015] In some embodiments, in addition to the amino acid substitutions at positions 126 and / or 129 as described above, the amino acid substitutions further include substituting one, two, three, four, five, six, seven, eight, or nine amino acid residues among positions 168, 227, 228, 229, 230, 233, 235, 239, and 241 in the amino acid sequence of pyrrolysine aminoacyl-tRNA synthetase shown in SEQ ID NO:1.

[0016] In some preferred embodiments, in addition to the amino acid substitutions at positions 126 and / or 129 as described above, the amino acid substitutions further include substituting one, two, three, four, five, six, seven, eight, or nine amino acid residues among positions 168, 227, 228, 229, 230, 233, 235, 239, and 241 in the amino acid sequence of pyrrolysine aminoacyl-tRNA synthetase shown in SEQ ID NO:1, and the amino acid substitutions are as follows:

[0017] Substituting the valine at position 168 with arginine, methionine, alanine, threonine, glycine, or cysteine;

[0018] Substituting the histidine at position 227 with asparagine, tyrosine, phenylalanine, or threonine;

[0019] Substituting the tyrosine at position 228 with phenylalanine, proline, or tryptophan;

[0020] Substituting the leucine at position 229 with glycine, alanine, valine, or isoleucine;

[0021] Substituting the aspartic acid at position 230 with tyrosine or phenylalanine;

[0022] Substituting the histidine at position 233 with glycine, valine, leucine, or isoleucine;

[0023] Substituting the valine at position 235 with lysine, glutamine, threonine, leucine, glycine, or isoleucine;

[0024] Substituting the tryptophan at position 239 with proline, phenylalanine, or arginine;

[0025] Substituting the glycine at position 241 with serine, cysteine, or leucine.

[0026] In some embodiments, the pyrrolysine aminoacyl-tRNA synthetase mutant comprises or consists of the amino acid sequences shown in SEQ ID NO:2 to SEQ ID NO:26.

[0027] In a second aspect, the present invention provides a nucleic acid molecule encoding a pyrrolysine aminoacyl-tRNA synthetase mutant.

[0028] In some embodiments, the nucleic acid molecule comprises or consists of a sequence as shown in SEQ ID NOs: 48 to 72.

[0029] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention, which is capable of producing the pyrrolysine aminoacyl-tRNA synthetase mutant of the present invention in a host cell when introduced into the host cell.

[0030] In a fourth aspect, the present invention provides a host cell transformed with the expression vector of the present invention.

[0031] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. Examples of the prokaryotic cell may be Escherichia coli of the genus Escherichia, Bacillus subtilis of the genus Bacillus, Pseudomonas putida of the genus Pseudomonas, etc. Examples of the eukaryotic cell may be yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc., and mammalian cells such as COS cells, CHO cells, HEK293 cells, BHK cells, etc. The transformation method can be carried out by known methods such as the method using calcium ions, the DEAE dextran method, the electroporation method, etc.

[0032] In a fifth aspect, the present invention provides the use of the pyrrolysine aminoacyl-tRNA synthetase mutant of the present invention in the preparation of a recombinant protein containing a non-natural amino acid.

[0033] In some embodiments, the pyrrolysine aminoacyl-tRNA synthetase mutant introduces a non-natural amino acid into a protein to obtain a recombinant protein containing the non-natural amino acid.

[0034] In some preferred embodiments, the pyrrolysine aminoacyl-tRNA synthetase mutant introduces a non-natural amino acid into a specific site of a protein.

[0035] In some preferred embodiments, the pyrrolysine aminoacyl-tRNA synthetase mutant specifically introduces the non-natural amino acid NPAK into a specific position of a protein, and the structural formula of the NPAK is as shown in formula (I):

[0036]

[0037] In some embodiments, the recombinant protein is recombinant human IL-2, whose amino acid sequence is shown in SEQ ID NO:32 and which contains the unnatural amino acid NPAK at position 45; preferably, the cysteine at position 125 of recombinant human IL-2 is replaced with serine.

[0038] In a sixth aspect, the present invention provides a method for preparing a recombinant protein containing an unnatural amino acid, which comprises: expressing a protein-encoding gene in a cell or a cell extract in the presence of the pyrrolysine aminoacyl-tRNA synthetase mutant and MatRNA to obtain a recombinant protein containing an unnatural amino acid.

[0039] In some preferred embodiments, the unnatural amino acid is introduced into a specific site of the recombinant protein, and in the protein-encoding gene, the codon corresponding to the specific site is replaced with an amber codon.

[0040] In some preferred embodiments, the sequence of the MatRNA is shown in SEQ ID NO:28.

[0041] In some preferred embodiments, NPAK is introduced into a specific site of the recombinant protein, and in the protein-encoding gene, the codon corresponding to the specific site is replaced with an amber codon; the structural formula of the NPAK is shown in formula (I):

[0042]

[0043] In some embodiments, the recombinant protein is a protein that can be used as a drug.

[0044] In some embodiments, the recombinant protein is recombinant human IL-2, whose amino acid sequence is shown in SEQ ID NO:32 and which contains the unnatural amino acid NPAK at position 45; preferably, the cysteine at position 125 of recombinant human IL-2 is replaced with serine.

[0045] By simulating and calculating the molecular structures of MaPylRS and NPAK, the present invention designed and constructed a molecular library of tens of millions of MaPylRS mutants, and obtained multiple NPAK-specific pyrrolysyl-tRNA synthetase mutants through high-throughput multi-step screening. Compared with the existing wild-type pyrrolysyl-tRNA synthetase (MbPylRS), the pyrrolysyl-tRNA synthetase mutants provided by the present invention have a more efficient recognition and catalytic efficiency for non-natural amino acids, especially NPAK, enabling host cells (such as Escherichia coli or mammalian cells) expressing the mutants to highly express recombinant proteins with NPAK, with the expression efficiency increased by 230% - 380%, which plays an important role in reducing the production cost of recombinant proteins and realizing the large-scale commercial production of recombinant proteins. Brief Description of the Drawings

[0046] Figure 1 It is the map of the expression vector of methanogenic pyrrolysyl-tRNA synthetase (MaPylRS) in Example 1 of the present invention;

[0047] Figure 2 It is the schematic diagram of the activity verification report plasmids pQ4-EGFP150*-MatRNA and pQ4-EGFP150*-MbtRNA of MaPylRS mutants and MbPylRS in Example 3 of the present invention;

[0048] Figure 3 It shows the relative fluorescence intensity of prokaryotic cells with MaPylRS mutants and MbPylRS in Example 3 of the present invention;

[0049] Figure 4 It is the SDS-PAGE electrophoresis diagram of the fermentation product obtained by the rhIL-2 expression strain after adding non-natural amino acid (NPAK) in Example 4 of the present invention;

[0050] Figure 5 It is the mass spectrum diagram of rhIL-2 with NPAK introduced at the 45th position in Example 4 of the present invention;

[0051] Figure 6 It is the fluorescence diagram of eukaryotic cells with MaPylRS mutants and MbPylRS in Example 5 of the present invention. Detailed Embodiments

[0052] The present invention will be further described below through specific examples. The examples described in the present invention are only for the illustration of the present invention and do not limit the scope of the present invention.

[0053] The present invention has obtained an efficient pyrrolysyl - tRNA synthetase mutant targeting NPAK through reasonable design and high - throughput screening, and has utilized this mutant to achieve efficient insertion of NPAK at specific protein sites in mammalian cells and Escherichia coli. The following further illustrates the present invention through specific experimental operations.

[0054] First step: Select at least one or a combination of amino acid residues from the amino acid sequences at positions 126, 129, 168, 227, 228, 229, 230, 233, 235, 239, and 241 of the wild - type pyrrolysyl - tRNA synthetase (MaPylRS, SEQ ID NO:1) of Methanosarcina barkeri as mutation sites, and construct a mutant library based on this.

[0055] The mutation sites are selected from the amino acid residues in the wild - type MaPylRS amino acid sequence that interact with the substrate pyrrolysine (Pyl).

[0056] Second step: Screen the MaPylRS mutant library using the unnatural amino acid NPAK and obtain a specific and efficient mutant MaPylRS.

[0057] Third step: Use the mutant MaPylRS and the corresponding MatRNA to express the target protein containing the unnatural amino acid NPAK in Escherichia coli and mammalian cells.

[0058] Example 1: Construction of an aminoacyl - tRNA synthetase mutant library

[0059] (1) Construction of plasmid pQ3 - MaPylRS containing the wild - type pyrrolysyl - tRNA synthetase (MaPylRS) of Methanosarcina barkeri:

[0060] The nucleotide sequence encoding MaPylRS is as shown in SEQ ID NO:27. The gene sequence was commissioned to be synthesized by Genewiz (Suzhou) Co., Ltd., cloned by PCR, and inserted into the pBK - JYRS plasmid backbone (Proc Natl Acad Sci USA. 2002 Aug 20; 99(17):11020 - 4) using NdeI / PstI enzymes to construct plasmid pQ3 - MaPylRS. The plasmid map is as Figure 1 . This plasmid mainly consists of a GlnRS promoter, MaPylRS, a GlnRS terminator, a ColE1 replication origin, and a kanamycin sulfate (Kan) resistance gene.

[0061] The cloning primers are as follows:

[0062] Ma1-F: gtttacgctttgaggaatcccatatgactgtaaaatatac (SEQ ID NO:34)

[0063] Ma1-R: tttagcgtttgaaactgcagttagttgatcttcgcgccgt (SEQ ID NO:35)

[0064] (2) Construction of the MaPylRS mutant library

[0065] Using the pQ3-MaPylRS plasmid as a template, a mutant library was constructed with degenerate primer NNK. In the first round, a library fragment containing a saturated mutation at the first site was amplified by PCR, and then the library fragment was used to replace the wild-type MaPylRS fragment in the template plasmid by enzymatic digestion and ligation. In the second round of amplification, the plasmid mutated in the previous round was used as a template for amplification and replacement. After 5 rounds (the mutation sites in the 5 rounds were 168, 126 + 129, 227 + 228 + 229 + 230, 233 + 235, 239 + 241 in sequence), the mutated positions in the library gradually increased, and finally a library containing all the designed mutated positions (the 126th, 129th, 168th, 227th, 228th, 229th, 230th, 233rd, 235th, 239th, and 241st positions of MaPylRS) was prepared. In theory, each mutated position contains 32 coding possibilities. 1000 clones were randomly selected from the library and verified by sequencing. The MaPylRS mutant sequences of all clones were different, and thus the library diversity was determined to be qualified. The final library plasmid containing all MaPylRS mutants was named pMa.

[0066] (The library construction method can be referred to ChemBioChem. 2013 Nov 4; 14(16): 2100 - 5).

[0067] Example 2: Screening of pyrrolysyl - tRNA synthetase mutants with site - directed introduction of NPAK

[0068] In this example, the MaPylRS random saturated mutant library plasmid pMa constructed in Example 1 was co - transformed with the positive - screening plasmid pREP - MatRNA Pyl into Escherichia coli DH10B for positive screening, and then co - transformed with the negative - screening plasmid pYOBB2 - MatRNA Pyl into Escherichia coli DH10B for negative screening (the principles of materials and methods can be referred to ACS Chem Biol. 2022 Dec 16; 17(12): 3458 - 3469). The specific steps are as follows:

[0069] (1) Construct Escherichia coli DH10B positive selection competent cells containing the screening plasmid pREP-MatRNA Pyl (carrying the tetracycline (Tet) resistance gene, the MatRNA gene, the chloramphenicol resistance gene (CAT) containing the amber stop codon (TAG), and the green fluorescent protein gene (GFP) containing the amber stop codon (TAG)), and transform the screening plasmid pREP-MatRNA Pyl into DH10B chemically competent cells, spread them on a 12.5 μg / ml Tet-resistant plate and culture overnight in a 37°C incubator. Inoculate them into 50 mL of 12.5 μg / mL Tet-resistant LB medium and culture overnight in a 37°C / 220 rpm constant temperature shaker. The next day, expand the culture 1:10 to 1 L of 2×YT and culture at 37°C / 220 rpm until the OD600 reaches about 0.5. Centrifuge at 4000×g, 4°C for 10 min and discard the supernatant. Add 500 mL of 4°C pre-cooled 10% glycerol solution and resuspend thoroughly. Centrifuge at 4000×g, 4°C for 10 min and discard the supernatant. Repeat this process once. Finally, aliquot the cells into 100 μL portions, quickly freeze them in liquid nitrogen and store them at -80°C, which are the pREP-MatRNA Pyl -DH10B electrocompetent cells.

[0070] (2) Screening of MaPylRS mutants. Electroporate the constructed pMa library plasmid into pREP-MatRNA Pyl -DH10B electrocompetent cells. Transfer all the bacterial solution and spread it on a Kan (kanamycin, 50 μg / mL), Tet (tetracycline, 25 μg / mL), Cm (chloramphenicol, 50 μg / mL), NPAK (1 mM), L-arabinose (0.2%) LB solid medium plate. Incubate it upside down in a 37°C constant temperature incubator for 48 h, collect the bacteria (MaPylRS mutants that recognize NPAK and natural amino acids, can read through the CAT gene with TAG and produce chloramphenicol resistance), and extract the plasmid.

[0071] (3) Construct Escherichia coli DH10B negative selection competent cells containing the screening plasmid pYOBB2-MatRNA Pyl (carrying the chloramphenicol (Cm) resistance gene, the MatRNA gene, and the barnase toxin protein gene containing the amber stop codon (TAG)) as described above. Electroporate the pMa library plasmid that has undergone one round of positive screening into pYOBB2-MatRNA PylIn -DH10B electrocompetent cells, spread them on LB solid medium plates containing 25 μg / mL Cm, 50 μg / mL Kan and 0.2% arabinose, place them upside - down in a 37°C incubator and culture overnight. Collect the bacteria (the MaPylRS mutants carried only recognize NPAK, otherwise the barnase toxin protein gene will be read through and the bacteria will die), and extract the plasmid.

[0072] The pMa library plasmid was repeatedly screened by positive and negative alternation for multiple rounds. Finally, 65 candidate clones were selected on the positive - screening plates and all were sequenced and analyzed. Sequencing primers Ma6 - F: gacggcggctttgttgaata (SEQ ID NO:36); Ma6 - R: cgaagcggaattaattcgcg (SEQ ID NO:37). Summarizing the sequencing results, a total of 25 different aminoacyl - tRNA synthetase mutants that can specifically recognize NPAK were obtained, named RA1 - RA25 respectively, and their amino acid sequences are shown in SEQ ID NO:2 to SEQ ID NO:26.

[0073] Example 3: Testing the efficiency of MaPylRS mutants in the prokaryotic expression system

[0074] In this example, enhanced green fluorescent protein (EGFP) was used as the reporter gene. By detecting the ratio of the fluorescence value of EGFP with NPAK to the cell concentration OD600 in Escherichia coli DH10B, the improvement of the recognition activity of MaPylRS mutants compared to MbPylRS for NPAK in prokaryotic cells was reflected.

[0075] (1) Construct verification plasmids pQ4 - EGFP150* - MatRNA and pQ4 - EGFP150* - MbtRNA

[0076] Plasmids pQ4 - EGFP150* - MatRNA and pQ4 - EGFP150* - MbtRNA are as Figure 2As shown, it mainly includes the EGFP150* gene driven by the arabinose promoter (araBAD promoter), the MatRNA (SEQ ID NO:28) or MbtRNA (SEQ ID NO:29) gene driven by the proK promoter, and the chloramphenicol resistance gene. The amino acid sequence of the EGFP150* gene is as shown in SEQ ID NO:30, and the codon of its 150th amino acid (the site where the unnatural amino acid NPAK is introduced, represented by X) is replaced with the amber codon (TAG). The EGFP150* gene fragment was synthesized by Suzhou Genewiz Biotechnology Co., Ltd., amplified by PCR using primers EG-F / EG-R, digested with BglII / SalI, and ligated with T4 to the pEVOL-pAzF plasmid backbone (Addgene, catalog number #31186) to obtain plasmid pQ4-EGFP150*. Using the pQ4-EGFP150* plasmid as a template, the pQ4-EGFP150*-MatRNA and pQ4-EGFP150*-MbtRNA gene fragments were amplified by PCR using primers Mat-s / Mat-as and Mbt-s / Mbt-as respectively, and plasmids pQ4-EGFP150*-MatRNA and pQ4-EGFP150*-MbtRNA were obtained using the q5 site-directed mutagenesis kit (NEB, catalog number E0554).

[0077] The primers are as follows:

[0078] EG-F: ggaattaaccatggtgagcaaaggcgaagaactg (SEQ ID NO:38)

[0079] EG-R: caaaacagccaagcttttaatgatgatgatgatgatgtttgtacagttcatc (SEQ ID NO:39)

[0080] Mat-s: ctagccagcggggttcgacgccccggtctctcgccaaattcgaaaagcctgctcaac (SEQID NO:73)

[0081] Mat-as: gttttagagacccgctggtcgccggaccgtcccccaatgcggggcgcatc (SEQ ID NO:74)

[0082] Mbt-s: atttagagtccattcgatctacatgatcaggtttccaattcgaaaagcctgctcaac (SEQID NO:75)

[0083] Mbt-as: ccgttcagccgggttagattcccggggtttccgccaaatgcggggcgcatc (SEQ ID NO:76)

[0084] (2) Comparison of the activities of MaPylRS mutants and MbPylRS in recognizing the unnatural amino acid NPAK

[0085] In Example 2, the MaPylRS mutants RA1 - RA25 screened were co - transfected with pQ4 - EGFP150* - MatRNA into Escherichia coli DH10B respectively, and MbPylRS was co - transfected with pQ4 - EGFP150* - MbtRNA into Escherichia coli DH10B. 100 μl of each was spread on LB solid medium plates with 50 μg / mL Kan and 25 μg / mL Cm resistance, and incubated overnight in an inverted position in a 37 °C constant temperature incubator. The next day, the co - transfected strains were picked and inoculated into 3 mL of LB medium with 50 μg / mL Kan and 25 μg / mL Cm resistance, and incubated overnight in a 37 °C / 220 rpm constant temperature shaker. On the third day, they were respectively expanded 1:100 into 2 mL of LB medium with 50 μg / mL Kan and 25 μg / mL Cm resistance and cultured until OD600 ≈ 0.5. Then, an arabinose inducer and the unnatural amino acid NPAK were added. Among them, the experimental group: Kan (50 μg / mL), Cm (25 μg / mL), NPAK (1 mM), L - arabinose (0.15%); the blank group: Kan (50 μg / mL), Cm (25 μg / mL), L - arabinose (0.15%). They were incubated in a 37 °C / 220 rpm constant temperature shaker for 16 h. Finally, the cells of each group were washed three times with PBS, resuspended with the same volume, 200 μl was taken to measure the fluorescence value with a microplate reader (excitation wavelength 488 nm, emission wavelength 502 nm). At the same time, each cell was diluted 1:10 with PBS, and the OD600 of the corresponding bacterial solution was measured with a Nanodrop 2000. The fluorescence value was divided by OD600 to obtain the relative fluorescence ratio (as Figure 3 shown).

[0086] The results showed that the recognition activities of MaPylRS mutants RA1 - RA25 for the unnatural amino acid NPAK were 2.3 - 3.8 times higher than those of MbPylRS used in the prior art in terms of the EGFP fluorescence ratio index. It can be seen that the MaPylRS mutants screened in the present invention have significantly improved recognition activities for NPAK in Escherichia coli.

[0087] Example 4: Testing the orthogonality and specificity of MaPylRS mutants

[0088] In this example, recombinant human IL-2 was used as the reporter gene, and recombinant human IL-2 containing NPAK was expressed and purified in Escherichia coli DH10B. Mass spectrometry identification was used to verify that the MaPylRS mutant specifically introduced NPAK into the medicinal protein.

[0089] The mature amino acid sequence of Homo sapiens interleukin 2 (as shown in SEQ ID NO: 31) was obtained from the National Center for Biotechnology Information database of the United States. The codon for tyrosine (Y) at position 45 of the amino acid sequence was mutated to the amber codon (TAG) for inserting unnatural amino acids, and the cysteine codon at position 125 was mutated to a serine codon (the cysteine at position 125 does not participate in the formation of disulfide bonds, but instead interferes with the formation of normal disulfide bonds during the renaturation process of the protein inclusion bodies of recombinant Homo sapiens IL-2. After mutation, the renaturation efficiency can be improved without significantly affecting its activity). To express the recombinant protein in Escherichia coli, methionine (Met) was added to the N-terminus of the protein sequence to initiate protein translation, and finally the amino acid sequence of recombinant human IL-2 (SEQ ID NO: 32, X represents an unnatural amino acid) was obtained. Then, the complete DNA sequence was synthesized by gene synthesis to obtain the gene sequence IL2-45* (SEQ ID NO: 33) of recombinant human IL-2.

[0090] (1) In this example, the RA2 mutant was selected as a representative, and the RA2 linearized gene fragment was obtained by PCR amplification using primers RA2-F / RA2-R, and cloned into the auxiliary plasmid pEVOL-pAcFRS.2.t1 (Addgene, catalog number #73544) using BglII / SalI, and finally the plasmid pEVOL-RA2 was obtained.

[0091] The cloning primers are as follows:

[0092] RA2-F: aggaggaattagatctatgactgtaaaatatacagatgc (SEQ ID NO: 40)

[0093] RA2-R: ttaaacgtcgacttagttgatcttcgcgc (SEQ ID NO: 41)

[0094] In this example, pBad-IL2-45* was used to express IL2 with NPAK. Construction of pBad-IL2-45*: The pBad / HisA (Invitrogen, catalog number V430-01) vector was double digested with NcoI / HindIII, and the linearized vector pBad-NcoI-HindII was obtained by gel extraction and recovery. The IL2-45* gene fragment synthesized by whole gene was amplified by PCR with primers IL2-F / IL2-R, double digested with NcoI / HindIII, and ligated to pBad-NcoI-HindII with T4 to obtain the target plasmid pBad-IL2-45*.

[0095] The cloning primers are as follows:

[0096] IL2-F: ggaattaaccatggcgcctacatccagctcga (SEQ ID NO:42)

[0097] IL2-R: aacagccaagcttttaggtcagcgtgctaataatg (SEQ ID NO:43)

[0098] (2) pEVOL-RA2 and pBad-IL2-45* plasmids were co-transformed into chemically competent Escherichia coli DH10B cells, spread on LB solid medium plates with 100 μg / mL Amp and 25 μg / mL Cm resistance, and cultured overnight in an inverted position in a 37°C constant temperature incubator. The co-transformed strains were picked, inoculated into 3 mL of LB medium with 100 μg / mL Amp and 25 μg / mL Cm resistance, and cultured overnight in a 37°C / 220 rpm constant temperature shaker. The next day, the strain was preserved and named IL2(NPAK)-10B.

[0099] The IL2(NPAK)-10B expression strain was inoculated into 1 L of 2×YT medium with 100 μg / mL Amp and 25 μg / mL Cm resistance, and cultured at 37°C / 220 rpm until the OD600 of the bacterial solution reached 0.8. In the experimental group, arabinose with a final concentration of 0.2% and 1 mM NPAK were added. At the same time, a negative group was set up with only arabinose with a final concentration of 0.2% added, and cultured at 37°C / 220 rpm for 8 hours for induced expression.

[0100] 1 mL of each of the above bacterial solutions was taken, centrifuged at 10000 rpm for 1 min, resuspended with PBS to an OD600 of 10, and SDS-PAGE electrophoresis was performed on each bacterial suspension. The SDS-PAGE electrophoresis pattern of the strain is shown in Figure 4 . Figure 4 The results showed that the expression strain could express the full-length target protein only when the unnatural amino acid NPAK was added.

[0101] Resuspend the collected bacterial cells above respectively with resuspension buffer (25 mM Tris, 6 mM EDTA, 1 mM DTT, pH 8.0), add 1% DNase (1 mg / mL), 0.5% PMSF, mix evenly, and homogenize 3 times under the pressure of 50 - 80 MPa with an ultra-high pressure homogenizer; centrifuge the homogenate at 10000 rpm for 20 min, and collect the crude inclusion bodies in the lower layer. Wash the obtained crude inclusion bodies twice with washing buffer (20 mM Tris-HCl, 100 mM NaCl, 2% TritonX-100, pH 8.0), and then wash once with ultrapure water to obtain purified inclusion bodies. Dissolve the purified inclusion bodies with denaturing buffer (20 mM Tris-HCl, 100 mM NaCl, 6 M guanidine hydrochloride, 1 mM DTT, pH 8.0), centrifuge at 10000 rpm after 30 min, and collect the supernatant as the denatured protein solution. Add 4 volumes of refolding buffer (20 mM Tris-HCl, 100 mM NaCl, pH 8.0) to the collected denatured protein solution, stir well and then let it stand for 12 h, centrifuge at 10000 rpm and collect the supernatant as the refolded protein solution. Concentrate the refolded protein solution to 1 / 4 of the original volume with an ultrafiltration membrane package (Millipore, Biomax-5) with a molecular weight cut-off of 5 kDa, exchange the buffer with replacement buffer (20 mM citric acid-sodium citrate buffer, pH 4.0), and further concentrate to a protein concentration of about 2.0 mg / mL, centrifuge at 10000 rpm and collect the supernatant, aliquot 1 mL and store at -80 °C, which is rhIL2-NPAK. Send the rhIL2-NPAK sample for liquid chromatography-mass spectrometry identification. The theoretical molecular weight of rhIL2-NPAK containing methionine is 15656.09, and the theoretical molecular weight of rhIL2-NPAK without methionine is 15524.82. The main component molecular weights identified by RP-HPLC liquid phase main peak mass spectrometry are 15656.25 and 15524.75 respectively, which are consistent with the theoretical molecular weights of rhIL2-NPAK containing methionine and rhIL2-NPAK without methionine (as Figure 5 shown).

[0102] As Figure 5 analyzed, the RA2 mutant can specifically introduce the unnatural amino acid NPAK into the designated position of the target protein, and cannot introduce natural amino acids into the target protein, showing good orthogonality.

[0103] Correspondingly, perform similar verifications on the RA1, RA3 - RA25 mutants respectively as in this example. The results show that these mutants can all specifically introduce the unnatural amino acid NPAK into the designated position of the target protein, and cannot introduce natural amino acids into the target protein, showing good orthogonality.

[0104] Example 5: Testing MaPylRS mutants in eukaryotic expression systems

[0105] In this example, EGFP was used as a reporter gene, and the expression level of EGFP with NPAK in eukaryotic cells was reflected by detecting the fluorescence intensity, so as to compare and verify that the MaPylRS mutant has a higher recognition and catalytic efficiency for NPAK than MbPylRS used in the prior art in eukaryotic cells.

[0106] (1) In this example, the RA2 mutant was selected, and the RA2 linearized gene fragment was obtained by PCR amplification using primers RA2-F1 / RA2-R1, and cloned into the auxiliary plasmid pCMV-MbPylRS (Addgene, catalog number #91706) by homologous recombination (Novoprotein C112 kit) to obtain the plasmid pCMV-RA2.

[0107] The cloning primers are as follows:

[0108] RA2-F1: aactgcacggaagcttgccaccatgactgtaaaatatacagat (SEQ ID NO:44)

[0109] RA2-R1: agtcgaggctgatcagcgggtttagttgatcttcgcgccgtt (SEQ ID NO:45)

[0110] The construction process of the expression plasmid pCDNA-EGFP150* was as follows: The pCDNA3.1(+) (GeneBank accession number #37680) vector was double digested with NotI and XhoI, and the linearized vector pCDNA3.1-NotI-XhoI was obtained by gel extraction and recovery. The EGFP150* gene fragment was amplified by PCR using primers EG-F1 / EG-R1 (the amino acid sequence of EGFP150* is shown in SEQ ID NO:30, and its 150th amino acid (the site where NPAK was introduced) was replaced with the amber codon (TAG), represented by X), digested with NotI / XhoI, and ligated to pCDNA3.1-BamHI-EcoRI with T4 to obtain the plasmid pCDNA3.1-EGFP150*.

[0111] The cloning primers are as follows:

[0112] EG-F1: cacagtggcggccgccaccatggtgagctagggcgaagaactg (SEQ ID NO:46)

[0113] EG-R1: tctagactcgagttatttgtacagttcatccataccga (SEQ ID NO:47)

[0114] The Chinese hamster ovary cell CHO-K1 (product number #CCL-61-ATC) was purchased from the American Type Culture Collection (ATCC) and cultured adherently using RPMI 1640 medium containing 10% fetal bovine serum. The auxiliary plasmids pCMV-MbPylRS and pCMV-RA2 in the above steps were extracted using an endotoxin-free plasmid extraction kit, and then mixed with the green fluorescent protein expression plasmid pCDNA3.1-EGFP150* respectively. The lipo2000 transfection reagent (Invitrogen, product number #12566014) was used for transient co-transfection according to the instructions (the cells were seeded into a 24-well plate at an inoculation density of 50,000 cells / well, and transfected 24 h after inoculation and culture, 500 ng of plasmid per well), and 1 mM NPAK at a final concentration was added to the experimental groups 2 h after transfection. The negative control group was not added with unnatural amino acids. After static culture in a carbon dioxide incubator for 48 h, observations and photographs were taken under a fluorescence microscope. The results showed that the green fluorescence of the experimental group transfected with the RA2 plasmid was significantly stronger than that of the cells transfected with the MbPylRS plasmid (as Figure 6 shown). It can be seen from this that compared with MbPylRS in eukaryotic cells, the MaPylRS mutant has a significantly improved recognition activity for NPAK.

[0115] Correspondingly, similar verifications were carried out on the RA1, RA3-RA25 mutants respectively. The results showed that compared with MbPylRS in eukaryotic cells, the recognition activities of these mutants for NPAK were significantly improved.

[0116] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

[0117] Sequence information:

[0118] Wild-type methanogenic methylotrophic pyrrolysyl-tRNA synthetase (SEQ ID NO:1)

[0119] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0120] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNL Y SV M RDLRDHT

[0121] DGPVKIFEMGSCFRKESHSGMHLEEFTMLNL V DMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEES

[0122] DVYKETIDVEINGQEVCSAAVGP HYLD AA H D V HEP W S G AGFGLERLLTIREKYSTVKKGGASISYLNG

[0123] AKIN

[0124] RA1 mutant (SEQ ID NO:2)

[0125] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0126] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0127] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0128] VYKETIDVEINGQEVCSAAVGPNPGYAAGDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0129] KIN

[0130] RA2 mutant (SEQ ID NO:3)

[0131] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0132] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0133] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLRDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0134] VYKETIDVEINGQEVCSAAVGPNPGYAAGDQHEPFSCAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0135] IN

[0136] RA3 mutant (SEQ ID NO:4)

[0137] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0138] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0139] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0140] VYKETIDVEINGQEVCSAAVGPYPGFAAGDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0141] KIN

[0142] RA4 mutant (SEQ ID NO:5)

[0143] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0144] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0145] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLTDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0146] VYKETIDVEINGQEVCSAAVGPTPGYAAGDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0147] KIN

[0148] RA5 mutant (SEQ ID NO:6)

[0149] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0150] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0151] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLRDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0152] VYKETIDVEINGQEVCSAAVGPNFLYAAVDQHEPFSCAGFGLERLLTIREKYSTVKKGGASISYLNGAKI

[0153] N

[0154] RA6 mutant (SEQ ID NO:7)

[0155] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0156] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0157] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0158] VYKETIDVEINGQEVCSAAVGPYFLFAAVDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0159] IN

[0160] RA7 mutant (SEQ ID NO:8)

[0161] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0162] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0163] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLRDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0164] VYKETIDVEINGQEVCSAAVGPNPGYAAGDKHEPPSSAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0165] IN

[0166] RA8 mutant (SEQ ID NO:9)

[0167] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0168] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0169] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLMDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0170] VYKETIDVEINGQEVCSAAVGPNWIYAAVDVHEPRSGAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0171] IN

[0172] RA9 mutant (SEQ ID NO:10)

[0173] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0174] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0175] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLTDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0176] VYKETIDVEINGQEVCSAAVGPFWLFAAGDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0177] KIN

[0178] RA10 mutant (SEQ ID NO:11)

[0179] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0180] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0181] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLRDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0182] VYKETIDVEINGQEVCSAAVGPNWVYAAGDKHEPPSSAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0183] KIN

[0184] RA11 mutant (SEQ ID NO:12)

[0185] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0186] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0187] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0188] VYKETIDVEINGQEVCSAAVGPNPGYAAGDTHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0189] KIN

[0190] RA12 mutant (SEQ ID NO:13)

[0191] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0192] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0193] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLADMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0194] VYKETIDVEINGQEVCSAAVGPNWAYAALDLHEPRSLAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0195] IN

[0196] RA13 mutant (SEQ ID NO:14)

[0197] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0198] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0199] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0200] VYKETIDVEINGQEVCSAAVGPNPGYAAGDGHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0201] KIN

[0202] RA14 mutant (SEQ ID NO:15)

[0203] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0204] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0205] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLTDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0206] VYKETIDVEINGQEVCSAAVGPNWAYAAIDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0207] KIN

[0208] RA15 mutant (SEQ ID NO:16)

[0209] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0210] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0211] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLCDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0212] VYKETIDVEINGQEVCSAAVGPNPGYAAGDTHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0213] KIN

[0214] RA16 mutant (SEQ ID NO:17)

[0215] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0216] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0217] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0218] VYKETIDVEINGQEVCSAAVGPNFVYAAIDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0219] IN

[0220] RA17 mutant (SEQ ID NO:18)

[0221] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0222] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0223] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0224] VYKETIDVEINGQEVCSAAVGPNFIYAALDGHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0225] IN

[0226] RA18 mutant (SEQ ID NO:19)

[0227] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0228] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0229] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLTDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0230] VYKETIDVEINGQEVCSAAVGPTWGYAAVDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0231] KIN

[0232] RA19 mutant (SEQ ID NO:20)

[0233] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0234] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0235] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLCDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0236] VYKETIDVEINGQEVCSAAVGPNWLYAAVDTHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGA

[0237] KIN

[0238] RA20 mutant (SEQ ID NO:21)

[0239] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0240] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0241] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLTDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0242] VYKETIDVEINGQEVCSAAVGPFPGFAAGDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0243] IN

[0244] RA21 mutant (SEQ ID NO:22)

[0245] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0246] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0247] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0248] VYKETIDVEINGQEVCSAAVGPNFAYAAVDTHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0249] IN

[0250] RA22 mutant (SEQ ID NO:23)

[0251] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0252] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0253] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLGDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0254] VYKETIDVEINGQEVCSAAVGPNFAYAAGDIHEPRSGAGFGLERLLTIREKYSTVKKGGASISYLNGAKI

[0255] N

[0256] RA23 mutant (SEQ ID NO:24)

[0257] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0258] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0259] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLMDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0260] VYKETIDVEINGQEVCSAAVGPNPGYAAGDVHEPRSGAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0261] IN

[0262] RA24 mutant (SEQ ID NO:25)

[0263] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0264] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0265] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLGDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0266] VYKETIDVEINGQEVCSAAVGPNPGYAAGDIHEPRSGAGFGLERLLTIREKYSTVKKGGASISYLNGAKI

[0267] N

[0268] RA25 mutant (SEQ ID NO:26)

[0269] MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLM

[0270] NDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLASVLRDLRDHTD

[0271] GPVKIFEMGSCFRKESHSGMHLEEFTMLNLADMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESD

[0272] VYKETIDVEINGQEVCSAAVGPNPGYAAGDLHEPRSLAGFGLERLLTIREKYSTVKKGGASISYLNGAK

[0273] IN

[0274] Nucleotide sequence encoding MaPylRS (SEQ ID NO:27)

[0275] atgactgtaaaatatacagatgctcaaatacaacgtctgcgcgaatatggtaatggcacctatgaacaaaaagtgttcgaggatcttgcgagccgcgacgctgcgtt

[0276] tagcaaggagatgagcgtcgcttctactgacaacgaaaagaagatcaaaggcatgattgccaatccgagccgtcatggtttgacgcagctgatgaatgatattgc

[0277] ggatgcgttggtggcggagggtttcatcgaagttcgtaccccgatctttatctcaaaggacgccctggcccgtatgacgatcaccgaagacaaaccgttatttaaac

[0278] aggttttctggattgacgaaaagcgcgcactgcgtccgatgctggctccgaatttgtattctgtcatgcgcgacctcagggatcacaccgatggtccggttaagatct

[0279] ttgaaatgggcagctgctttcgtaaagagagccactccggcatgcatctggaggagttcaccatgctgaacctggtggacatgggtccgcgtggtgatgctaccg

[0280] aggttctgaaaaactatatctctgtggtgatgaaagcggcgggtctgccggattacgacttggttcaggaggagtccgatgtctacaaagagacaatcgacgtgga

[0281] aattaacggccaagaggtgtgttccgcagcggttggtcctcactatctcgatgcggcgcacgatgtgcatgaaccgtggagcggtgcaggcttcggcctggaac

[0282] gtttgttgaccattcgtgagaagtacagcaccgttaaaaagggtggtgcaagcatttcgtacctgaacggcgcgaagatcaactaa

[0283] MatRNA(SEQ ID NO:28)

[0284] gggggacggtccggcgaccagcgggtctctaaaacctagccagcggggttcgacgccccggtctctcgcca

[0285] MbtRNA(SEQ ID NO:29)

[0286] tggcggaaaccccgggaatctaacccggctgaacggatttagagtccattcgatctacatgatcaggtttcc

[0287] EGFP150*(SEQ ID NO:30)

[0288] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGV

[0289] QCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI

[0290] LGHKLEYNYNSHXVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST

[0291] QSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH

[0292] Homo sapiens mature IL-2(SEQ ID NO:31)

[0293] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV

[0294] LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF C QSIISTLT

[0295] Recombinant human IL-2(SEQ ID NO:32)

[0296] MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFXMPKKATELKHLQCLEEELKPLEE

[0297] VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF S QSIISTLT

[0298] Gene sequence of recombinant human IL-2 (SEQ ID NO:33)

[0299] atggcgcctacatccagctcgaccaaaaagactcaactgcaactggaacacctgctcctggatctgcaaatgattcttaacggtatcaataactacaaaaatccgaa

[0300] actgacccgtatgctgacgtttaaattctagatgccaaagaaagcgaccgagctgaaacatctgcagtgcctggaagaggaactgaaaccgctggaggaagtttt

[0301] gaacctggctcagtctaaaaactttcacctgcgccctcgtgacctgatttccaatatcaacgtgattgttctggaactgaaaggctctgaaaccacgtttatgtgcgag

[0302] tacgccgatgaaaccgccacgattgtggaatttctgaatcgctggatcaccttctcccagagcattattagcacgctgacctaa

[0303] Nucleotide sequence encoding RA1 (SEQ ID NO:48)

[0304] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0305] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0306] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0307] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0308] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0309] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0310] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0311] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGTAGACATGGGTCCGCGTGGTGATGCT

[0312] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0313] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0314] TTCCGCAGCGGTTGGTCCTAATCCGGGTTATGCGGCGGGTGATGTACATGAACCGTGGAGCGGAGC

[0315] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0316] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0317] Nucleotide sequence encoding RA2 (SEQ ID NO:49)

[0318] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0319] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0320] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0321] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0322] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0323] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0324] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0325] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGCGTGACATGGGTCCGCGTGGTGATGCT

[0326] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0327] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0328] TTCCGCAGCGGTTGGTCCTAATCCGGGTTATGCGGCGGGTGATCAGCATGAACCGTTCAGCTGCGC

[0329] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0330] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0331] Nucleotide sequence encoding RA3 (SEQ ID NO:50)

[0332] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0333] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0334] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0335] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0336] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0337] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0338] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0339] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGTAGACATGGGTCCGCGTGGTGATGCT

[0340] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0341] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0342] TTCCGCAGCGGTTGGTCCTTATCCGGGTTTCGCGGCGGGTGATGTGCATGAACCGTGGAGCGGAGC

[0343] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0344] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0345] Nucleotide sequence encoding RA4 (SEQ ID NO:51)

[0346] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0347] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0348] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0349] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0350] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0351] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0352] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0353] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGACCGACATGGGTCCGCGTGGTGATGCT

[0354] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0355] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0356] TTCCGCAGCGGTTGGTCCTACGCCGGGTTACGCGGCGGGTGATGTCCATGAACCGTGGAGCGGCGC

[0357] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0358] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0359] Nucleotide sequence encoding RA5 (SEQ ID NO:52)

[0360] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0361] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0362] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0363] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0364] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0365] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0366] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0367] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGCGCGACATGGGTCCGCGTGGTGATGCT

[0368] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0369] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0370] TTCCGCAGCGGTTGGTCCTAATTTCCTGTACGCGGCGGTAGATCAACATGAACCGTTCAGCTGCGC

[0371] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0372] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0373] Nucleotide sequence encoding RA6 (SEQ ID NO:53)

[0374] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0375] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0376] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0377] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0378] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0379] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0380] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0381] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGTAGACATGGGTCCGCGTGGTGATGCT

[0382] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0383] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0384] TTCCGCAGCGGTTGGTCCTTACTTCTTATTCGCGGCGGTCGATGTACATGAACCGTGGAGCGGAGC

[0385] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0386] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0387] Nucleotide sequence encoding RA7 (SEQ ID NO:54)

[0388] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0389] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0390] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0391] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0392] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0393] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0394] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0395] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGCGTGACATGGGTCCGCGTGGTGATGCT

[0396] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0397] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0398] TTCCGCAGCGGTTGGTCCTAATCCGGGTTATGCGGCGGGTGATAAGCATGAACCGCCGAGCAGCGC

[0399] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0400] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0401] Nucleotide sequence encoding RA8 (SEQ ID NO:55)

[0402] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0403] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0404] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0405] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0406] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0407] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0408] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0409] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGATGGACATGGGTCCGCGTGGTGATGCT

[0410] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0411] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0412] TTCCGCAGCGGTTGGTCCTAATTGGATCTACGCGGCGGTAGATGTGCATGAACCGCGCAGCGGAGC

[0413] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0414] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0415] Nucleotide sequence encoding RA9 (SEQ ID NO:56)

[0416] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0417] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0418] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0419] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0420] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0421] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0422] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0423] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGACAGACATGGGTCCGCGTGGTGATGCT

[0424] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0425] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0426] TTCCGCAGCGGTTGGTCCTTTCTGGCTCTTCGCGGCGGGCGATGTACATGAACCGTGGAGCGGAGC

[0427] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0428] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0429] Nucleotide sequence encoding RA10 (SEQ ID NO:57)

[0430] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0431] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0432] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0433] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0434] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0435] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0436] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0437] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGCGCGACATGGGTCCGCGTGGTGATGCT

[0438] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0439] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0440] TTCCGCAGCGGTTGGTCCTAATTGGGTATACGCGGCGGGAGATAAACATGAACCGCCAAGCAGCGC

[0441] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0442] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0443] Nucleotide sequence encoding RA11 (SEQ ID NO:58)

[0444] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0445] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0446] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0447] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0448] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0449] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0450] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0451] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGTAGACATGGGTCCGCGTGGTGATGCT

[0452] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0453] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0454] TTCCGCAGCGGTTGGTCCTAATCCGGGTTATGCGGCGGGTGATACACATGAACCGTGGAGCGGAGC

[0455] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0456] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0457] Nucleotide sequence encoding RA12 (SEQ ID NO:59)

[0458] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0459] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0460] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0461] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0462] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0463] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0464] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0465] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGCAGACATGGGTCCGCGTGGTGATGCT

[0466] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0467] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0468] TTCCGCAGCGGTTGGTCCTAATTGGGCATACGCGGCGCTAGATCTGCATGAACCGAGAAGCCTCGC

[0469] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0470] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0471] Nucleotide sequence encoding RA13 (SEQ ID NO:60)

[0472] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0473] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0474] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0475] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0476] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0477] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0478] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0479] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGTCGACATGGGTCCGCGTGGTGATGCT

[0480] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0481] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0482] TTCCGCAGCGGTTGGTCCTAATCCGGGTTATGCGGCGGGTGATGGCCATGAACCGTGGAGCGGCGC

[0483] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0484] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0485] Nucleotide sequence encoding RA14 (SEQ ID NO:61)

[0486] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0487] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0488] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0489] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0490] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0491] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0492] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0493] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGACAGACATGGGTCCGCGTGGTGATGCT

[0494] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0495] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0496] TTCCGCAGCGGTTGGTCCTAATTGGGCATACGCGGCGATCGATGTACATGAACCGTGGAGCGGAGC

[0497] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0498] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0499] Nucleotide sequence encoding RA15 (SEQ ID NO:62)

[0500] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0501] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0502] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0503] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0504] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0505] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0506] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0507] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGTGCGACATGGGTCCGCGTGGTGATGCT

[0508] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0509] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0510] TTCCGCAGCGGTTGGTCCTAACCCGGGTTACGCGGCGGGTGATACCCATGAACCGTGGAGCGGCGC

[0511] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0512] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0513] Nucleotide sequence encoding RA16 (SEQ ID NO:63)

[0514] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0515] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0516] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0517] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0518] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0519] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0520] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0521] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGTAGACATGGGTCCGCGTGGTGATGCT

[0522] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0523] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0524] TTCCGCAGCGGTTGGTCCTAATTTCGTATATGCGGCGATCGATGTACATGAACCGTGGAGCGGAGCA

[0525] GGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTGC

[0526] AAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0527] Nucleotide sequence encoding RA17 (SEQ ID NO:64)

[0528] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0529] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0530] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0531] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0532] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0533] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0534] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0535] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGTAGACATGGGTCCGCGTGGTGATGCT

[0536] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0537] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0538] TTCCGCAGCGGTTGGTCCTAATTTCATCTACGCGGCGCTGGATGGTCATGAACCGTGGAGCGGAGC

[0539] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0540] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0541] Nucleotide sequence encoding RA18 (SEQ ID NO:65)

[0542] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0543] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0544] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0545] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0546] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0547] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0548] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0549] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGACAGACATGGGTCCGCGTGGTGATGCT

[0550] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0551] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0552] TTCCGCAGCGGTTGGTCCTACATGGGGATACGCGGCGGTAGATGTACATGAACCGTGGAGCGGAGC

[0553] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0554] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0555] Nucleotide sequence encoding RA19 (SEQ ID NO:66)

[0556] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0557] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0558] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0559] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0560] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0561] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0562] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0563] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGTGCGACATGGGTCCGCGTGGTGATGCT

[0564] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0565] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0566] TTCCGCAGCGGTTGGTCCTAATTGGCTATACGCGGCGGTAGATACACATGAACCGTGGAGCGGAGC

[0567] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0568] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0569] Nucleotide sequence encoding RA20 (SEQ ID NO: 67)

[0570] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0571] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0572] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0573] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0574] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0575] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0576] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0577] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGACTGACATGGGTCCGCGTGGTGATGCT

[0578] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0579] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0580] TTCCGCAGCGGTTGGTCCTTTCCCGGGTTTTGCGGCGGGTGATGTCCATGAACCGTGGAGCGGCGC

[0581] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0582] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0583] Nucleotide sequence encoding RA21 (SEQ ID NO:68)

[0584] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0585] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0586] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0587] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0588] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0589] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0590] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0591] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGTAGACATGGGTCCGCGTGGTGATGCT

[0592] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0593] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0594] TTCCGCAGCGGTTGGTCCTAATTTCGCATACGCGGCGGTAGATACCCATGAACCGTGGAGCGGAGC

[0595] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0596] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0597] Nucleotide sequence encoding RA22 (SEQ ID NO:69)

[0598] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0599] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0600] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0601] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0602] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0603] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0604] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0605] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGGCGACATGGGTCCGCGTGGTGATGCT

[0606] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0607] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0608] TTCCGCAGCGGTTGGTCCTAATTTCGCATACGCGGCGGGTGATATCCATGAACCGCGTAGCGGAGC

[0609] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0610] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0611] Nucleotide sequence encoding RA23 (SEQ ID NO:70)

[0612] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0613] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0614] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0615] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0616] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0617] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0618] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0619] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGATGGACATGGGTCCGCGTGGTGATGCT

[0620] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0621] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0622] TTCCGCAGCGGTTGGTCCTAATCCGGGTTATGCGGCGGGTGATGTCCATGAACCGCGGAGCGGAGC

[0623] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0624] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0625] Nucleotide sequence encoding RA24 (SEQ ID NO:71)

[0626] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0627] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0628] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0629] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0630] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0631] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0632] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0633] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGGAGACATGGGTCCGCGTGGTGATGCT

[0634] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0635] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0636] TTCCGCAGCGGTTGGTCCTAATCCGGGTTATGCGGCGGGTGATATCCATGAACCGAGAAGCGGCGC

[0637] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0638] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

[0639] Nucleotide sequence encoding RA25 (SEQ ID NO:72)

[0640] ATGACTGTAAAATATACAGATGCTCAAATACAACGTCTGCGCGAATATGGTAATGGCACCTATGAAC

[0641] AAAAAGTGTTCGAGGATCTTGCGAGCCGCGACGCTGCGTTTAGCAAGGAGATGAGCGTCGCTTCT

[0642] ACTGACAACGAAAAGAAGATCAAAGGCATGATTGCCAATCCGAGCCGTCATGGTTTGACGCAGCT

[0643] GATGAATGATATTGCGGATGCGTTGGTGGCGGAGGGTTTCATCGAAGTTCGTACCCCGATCTTTATC

[0644] TCAAAGGACGCCCTGGCCCGTATGACGATCACCGAAGACAAACCGTTATTTAAACAGGTTTTCTGG

[0645] ATTGACGAAAAGCGCGCACTGCGTCCGATGCTGGCTCCGAATTTGGCTTCTGTCCTGCGCGACCTC

[0646] AGGGATCACACCGATGGTCCGGTTAAGATCTTTGAAATGGGCAGCTGCTTTCGTAAAGAGAGCCAC

[0647] TCCGGCATGCATCTGGAGGAGTTCACCATGCTGAACCTGGCCGACATGGGTCCGCGTGGTGATGCT

[0648] ACCGAGGTTCTGAAAAACTATATCTCTGTGGTGATGAAAGCGGCGGGTCTGCCGGATTACGACTTG

[0649] GTTCAGGAGGAGTCCGATGTCTACAAAGAGACAATCGACGTGGAAATTAACGGCCAAGAGGTGTG

[0650] TTCCGCAGCGGTTGGTCCTAATCCGGGTTATGCGGCGGGTGATCTACATGAACCGCGCAGCTTGGC

[0651] AGGCTTCGGCCTGGAACGTTTGTTGACCATTCGTGAGAAGTACAGCACCGTTAAAAAGGGTGGTG

[0652] CAAGCATTTCGTACCTGAACGGCGCGAAGATCAAC

Claims

1. A pyrrolysine aminoacyl-tRNA synthetase mutant, wherein, The pyrrolysine aminoacyl-tRNA synthetase mutant is selected from one of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO:

26.

2. A nucleic acid molecule encoding the pyrrolysine aminoacyl-tRNA synthetase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein, The nucleic acid molecule comprises or consists of a sequence shown in any one of SEQ ID NO: 48 to SEQ ID NO:

72.

4. An expression vector comprising the nucleic acid molecule according to claim 2 or 3, which can produce the pyrrolysine aminoacyl-tRNA synthetase mutant according to claim 1 in a host cell when introduced into the host cell.

5. A host cell transformed with the expression vector according to claim 4.

6. The host cell according to claim 5, wherein, The host cell is a prokaryotic cell or a eukaryotic cell.

7. Use of the pyrrolysine aminoacyl-tRNA synthetase mutant according to claim 1 in the preparation of a recombinant protein containing a non-natural amino acid, wherein, The unnatural amino acid is NPAK, and its structural formula is shown in formula (I):

8. The use according to claim 7, characterized in that, The pyrrolysine aminoacyl-tRNA synthetase mutant introduces an unnatural amino acid into a protein to obtain a recombinant protein containing the unnatural amino acid.

9. The use according to claim 8, characterized in that, The pyrrolysine aminoacyl-tRNA synthetase mutant introduces an unnatural amino acid into a specific site of the protein.

10. A method for preparing a recombinant protein containing non-natural amino acids, comprising: In the presence of the pyrrolysine aminoacyl-tRNA synthetase mutant according to claim 1 and MatRNA, the coding gene of the protein is expressed in a cell or a cell extract to obtain a recombinant protein containing the unnatural amino acid; The sequence of the MatRNA is shown in SEQ ID NO: 28; The unnatural amino acid is NPAK, and its structural formula is shown in formula (I):

11. The preparation method according to claim 10, wherein The recombinant protein has the unnatural amino acid introduced at a specific site, and in the coding gene of the protein, the codon corresponding to the specific site is replaced with an amber codon.

12. The preparation method according to claim 10 or 11, characterized in that, The recombinant protein is a protein that can be used as a drug.

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