A method for preparing a mutant dihydropteridine synthetase with improved thermal stability

Abstract

The application relates to a preparation method of a dihydropteridine synthetase mutant with improved thermal stability. The preparation method comprises the following steps: obtaining an amino acid site of a receptor protein-DHPPP crystal complex affected by SMM; respectively performing single-point mutation on thermal stability and affinity of amino acids in a ligand; obtaining key amino acids of thermal stability of the receptor protein; determining mutated amino acids by simulating stability and affinity before and after mutation of the key amino acids through software; and constructing an amino acid sequence of the receptor protein with thermal stability based on the mutated amino acids. Biological experimental results show that the receptor protein mutant obtained by the preparation method has improved thermal stability, and the preparation method provides a theoretical basis for modification of thermal stability of the receptor protein.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of structural biology, bioinformatics and immunology, and particularly relates to a preparation method of a dihydropteroate synthase mutant with improved thermostability. BACKGROUND

[0002] The immunological analysis method is one of the commonly used methods for detecting sulfonamide residues at present, and its principle is based on the specific binding of antigen and antibody. The immunological analysis method has the characteristics of high sensitivity and high specificity, and has the advantages of high throughput, rapid detection, simple operation, and suitability for on-site screening, and has been widely used in sulfonamide residue screening.

[0003] The core reagent of the immunological analysis method is antibody. The conventional antibody preparation method mainly includes obtaining polyclonal serum by immunizing animals and obtaining monoclonal antibody by using hybridoma technology. Although the sensitivity of the antibody is high, the cycle for obtaining the antibody is long, the process is complex, the cost is high, and it is difficult to obtain an antibody that can recognize all sulfonamides. Therefore, the application of the immunological analysis method in the field of veterinary drug residue detection is restricted.

[0004] Receptor protein is a kind of broad-spectrum recognition material for drug molecules in the true sense. It is simple to prepare, can uniformly recognize a large class of drug molecules, and is one of the breakthroughs for solving the bottleneck problem of residue analysis technology. Dihydropteroate synthase (DHPS) is one of the catalytic enzymes for folate synthesis, and is the receptor protein of sulfonamides, which can recognize at least 28 kinds of sulfonamides. At present, there is a sulfonamide immunological analysis method based on DHPS. However, due to the poor thermal stability of DHPS, the detection of sulfonamide residues in actual samples is limited. Obtaining a DHPS mutant with improved thermal stability will help the detection of sulfonamides in actual samples. SUMMARY

[0005] Based on the defects in the prior art, the purpose of the present application is to provide a dihydropteroate synthase mutant with improved thermal stability and a preparation method thereof.

[0006] In a first aspect, the present application provides a dihydropteroate synthase mutant MDHPS, wherein the dihydropteroate synthase mutant MDHPS comprises that the ARG at the 238th position of the wild-type amino acid sequence is mutated to TRP, the GLU at the 245th position is mutated to CYS, the HIS at the 284th position is mutated to TRP, the SER at the 235th position is mutated to VAL, and the VAL at the 251st position is mutated to ILE.

[0007] Specifically, the amino acid sequence of the dihydropteroate synthase mutant MDHPS provided by the present application is shown in SEQ ID NO. 2.

[0008] MSSKANHAKTVICGIINVTPDSFGGQFFALEQALQQARKLIAEGASMLDIGGESTRPGSSYVEIEEEIQRVVPVIKAIRKESDVLISIDTWKSQVAEAALAAGADLVNDITGLMGDEKMPHVVAEARAQVVIMFNPVMARPQHPSSLIFPHFGFG QAFTEEELADFETLPIEELMEAFFERALARAAEAGIAPENILLDPGIGFGLTKKENLLLLRDLDKLHQKGYPIFLGVVRKWFVINILCENGFEINPETELGFRNRDTASAHVTSIAARQGVEVVRVWDVASHRMAVEIASAIRLADEAENLDLKQYK.

[0009] Secondly, the present invention provides the encoding gene of the above-mentioned dihydropteroate synthase mutant MDHPS, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] ATGAGCAGCAAAGCGAACCATGCAAAAACCGTAATTTGTGGTATTATTAACGTGACGCCGGATAGCTTTAGCGATGGTGGTCAGTTTTTTGCACTGGAACAGGCGCTGCAGCAGGCACGTAAACTGATCGCAGAAGGTGCATCCATGCTGGATATTGGTGGTGAAAGCACACGCCCTGGTTCAAGCTATGTTGAAATTGAAGAAGAAATCCAGCGTGTTGTTCCGGTTATTAAAGCAATTCGTAAAGAAAGCGATGTTCTGATTTCTATTGATACATGGAAATCACAGGTTGCAGAAGCAGCACTGGCGGCAGGTGCAGATCTGGTTAATGATATTACCGGTCTGATGGGTGATGAAAAAATGCCGCATGTTGTTGCAGAAGCACGTGCACAGGTTGTTATTATGTTTAATCCGGTTATGGCACGTCCGCAGCATCCGAGCAGCCTGATTTTTCCGCATTTTGGTTTTGGTCAGGCATTTACCGAAGAAGAACTGGCAGATTTTGAAACCCTGCCGATTGAAGAACTGATGGAAGCATTTTTTGAACGTGCACTGGCACGTGCAGCAGAAGCAGGTATTGCACCGGAAAATATTCTGCTGGATCCGGGTATTGGTTTTGGTCTGACCAAAAAAGAAAATCTGCTGCTGCTGCGTGATCTGGATAAACTGCATCAGAAAGGTTATCCGATTTTTCTGGGTGTTGTTCGTAAATGGTTTGTTATTAATATTCTGTGTGAAAATGGTTTTGAAATTAATCCGGAAACCGAACTGGGTTTTCGTAATCGTGATACCGCAAGCGCACATGTTACCAGCATTGCAGCACGTCAGGGTGTTGAAGTTGTTCGTGTTTGGGATGTTGCAAGCCATCGTATGGCAGTTGAAATTGCAAGCGCAATTCGTCTGGCAGATGAAGCAGAAAATCTGGATCTGAAACAGTATAAA。

[0011] Thirdly, the present invention provides a biological material that can express the above-mentioned dihydropteroate synthase mutant MDHPS or contains the above-mentioned coding gene; the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector, engineered bacteria or transgenic cell line.

[0012] Based on the understanding of those skilled in the art, this invention seeks protection for the application of the above-mentioned dihydropteroate synthase mutant MDHPS, or the above-mentioned encoding gene, or the above-mentioned biological material in the detection of sulfonamide drug residues.

[0013] Fourthly, the present invention provides a reagent or kit for detecting sulfonamide drug residues, wherein the reagent or kit uses the above-mentioned dihydropteroate synthase mutant MDHPS as the receptor protein for detecting sulfonamide drug residues.

[0014] Fifthly, the present invention provides a method for detecting sulfonamide drug residues, using the aforementioned dihydropteroate synthase mutant MDHPS as the immune receptor protein for detecting sulfonamide drug residues.

[0015] Sixthly, the present invention provides a method for preparing a thermostable receptor protein for detecting sulfonamide drug residues, comprising:

[0016] (1) Obtain the amino acid sites for the interaction between the receptor protein-DHPPP crystal complex and SMM;

[0017] (2) Ligands Single-point mutations were performed on the thermal stability and affinity of the amino acids within the specified range;

[0018] (3) Obtain the key amino acids for the thermostability of receptor proteins;

[0019] (4) The mutated amino acid was determined by simulating the stability and affinity of key amino acids before and after mutation using software.

[0020] (5) Based on the mutant amino acids, construct the amino acid sequence of the thermostable receptor protein.

[0021] In the preparation method provided by the present invention, the thermostable receptor protein is the dihydropteroate synthase mutant MDHPS.

[0022] Specifically, the present invention provides a method for improving the thermal stability of DHPS using technologies such as computer graphics, comprising the following steps:

[0023] Based on the PDB structural information database, the spatial structure information of the DHPS-DHPPP crystal complex (2VEG) of Streptococcus pneumoniae R6 was imported into Discovery Studio 2019 software;

[0024] Molecular docking was performed between the DHPS-DHPPP complex and SMM. The docking results showed that the key amino acids for the interaction between the DHPS-DHPPP complex and the sulfonamide drug SMM were located at: ARG212, ARG214, ARG238, GLU221, HIS260, ILE217, LYS213, PRO229, SER211, and VAL227.

[0025] Select the right ligand Single-point mutations were performed on the amino acids to determine thermal stability and affinity. This invention used Discovery Studio 2019 software to identify key amino acids affecting the thermal stability of receptor proteins. The amino acid sites corresponding to the highly thermally stable receptor protein DHPS are ARG238>TRP, GLU245>CYS, HIS284>TRP, SER235>VAL, and VAL251>ILE.

[0026] Therefore, the amino acid sequence of the dihydropteroate synthase mutant MDHPS provided by the present invention was obtained by mutating ARG at position 238 to TRP, GLU at position 245 to CYS, HIS at position 284 to TRP, SER at position 235 to VAL, and VAL at position 251 to ILE from the amino acid sequence of wild-type dihydropteroate synthase.

[0027] In the preparation method provided by the present invention, when the thermostable receptor protein is a dihydropteranoic acid synthase mutant MDHPS, the preparation of the thermostable receptor protein includes: the target fragment mdhps encoding the dihydropteranoic acid synthase mutant MDHPS is obtained by splicing the nucleotide sequences shown in SEQ ID NO.3-24 using overlap extension PCR technology, with an NheI restriction site inserted into the nucleotide sequence shown in SEQ ID NO.3 and an XhoI restriction site inserted into the nucleotide sequence shown in SEQ ID NO.24;

[0028] The expression vector pET-28a was double-digested with NdeI and XhoI. The target fragment was ligated into the expression vector to obtain the recombinant expression vector pET-28a-mdhps. The recombinant expression vector pET-28a-mdhps was transformed into Escherichia coli BL21(DE3) to obtain a recombinant expression strain that efficiently expresses recombinant Streptococcus pneumoniae dihydrofolate reductase.

[0029] OD in the expression strain culture system 600 When the concentration is 0.6-0.8, IPTG is added to the fermentation system and induced at 16-28℃ for 5-16 hours to obtain the dihydropteroate synthase mutant MDHPS.

[0030] The beneficial effects of this invention are as follows:

[0031] This invention provides a precise and efficient method for improving the thermal stability of receptor proteins. This invention utilizes the principles of computer graphics, structural biology, and immunology to obtain a thermostable dihydropteroate synthase mutant, MDHPS.

[0032] This invention theoretically explores the key factors affecting the thermal stability of receptor proteins, and combined with biological experiments, confirms that the method provided by this invention is applicable to obtaining receptor proteins with high thermal stability, providing a theoretical basis for modifying the thermal stability of receptor proteins. Attached Figure Description

[0033] Figure 1 This is the DHPS amino acid sequence of Streptococcus pneumoniae R6 in this invention.

[0034] Figure 2 This is a spatial model diagram of DHPS in this invention.

[0035] Figure 3 The diagram shows the DHPS amino acid sequence in the crystal structure of Streptococcus pneumoniae R6 in this invention.

[0036] Figure 4 This is a docking diagram of different amino acid mutations with SMM in this invention.

[0037] Figure 5 This is a two-dimensional interaction diagram of DHPS-SMM in this invention.

[0038] Figure 6 This is a PCR verification diagram of the mutant DNA fragment mdhps in this invention.

[0039] Figure 7 This is an SDS-PAGE image of the mutant MDHPS in this invention.

[0040] Figure 8 Thermostability diagrams of wild-type DHPS and mutant MDHPS. Detailed Implementation

[0041] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0042] Example 1

[0043] This invention first provides a method for improving the thermal stability of DHPS using technologies such as computer graphics. The method includes the following steps:

[0044] A. The DHPS amino acid sequence of Streptococcus pneumoniae R6 was obtained from GenBank in NCBI. Analysis using the ExPASy database showed that DHPS consists of 314 amino acids. Figure 1 This protein, with a molecular weight of approximately 35 kDa and a theoretical isoelectric point of 4.98, is an acidic protein that carries a negative charge in a neutral environment. The specific amino acid composition is shown in Table 1. It contains 47 negatively charged amino acids (Asp + Glu) and 31 positively charged amino acids (Arg + Lys), with a molar extinction coefficient of 9970 M. -1 cm -1 The instability coefficient in water is 40.38, indicating that the enzyme is an unstable protein.

[0045] Table 1. Amino acid composition of dihydropteroate synthase

[0046]

[0047] B. This invention uses PDB structural information database information to import the spatial structure information of the DHPS-DHPPP crystal complex (2VEG) of Streptococcus pneumoniae R6 into Discovery Studio 2019 software. Figure 2 The crystal amino acid sequence is as follows: Figure 3 Molecular docking was performed between the DHPS-DHPPP complex and SMM. The docking results showed that the key amino acids for interaction with SMM were located at: ARG212, ARG214, ARG238, GLU221, HIS260, ILE217, LYS213, PRO229, SER211, and VAL227.

[0048] C. Selecting the right ligand Single-point mutations were performed on the amino acids to assess their thermal stability and affinity. Figure 4 This invention uses Discovery Studio 2019 software to apply a CHARMm force field to DHPS-DHPPP, and then performs single-point mutations on the amino acids to identify the key amino acids affecting the thermal stability of the receptor protein: ARG212, ARG214, ARG238, GLU221, HIS260, ILE217, LYS213, PRO229, SER211, and VAL227.

[0049] D. Name the 10 amino acids Mutation and use the Calculate Mutation Energy (Stability) function in the DesignProtein module of Discovery Studio 2019 to mutate them into 19 other amino acids.

[0050] Table 2 lists the ligands. The mutation energy of single-point mutations in amino acids to improve stability and affinity.

[0051]

[0052]

[0053] Table 2 shows that when the crystal structure has the following parameters: ARG214>TRP (representing the mutation from ARG to TRP at position 214), GLU221>CYS, HIS260>TRP, SER211>VAL, and VAL227>ILE (these parameters represent amino acid sites in the crystal structure; the corresponding amino acid sites for the receptor protein DHPS are ARG238, GLU245, HIS284, SER235, and VAL251) Figure 5 The mutations at all five sites were less than 0.5, and the corresponding effects were stable. This indicates that mutations at these five sites can improve the thermostability of the receptor protein DHPS, while maintaining or slightly increasing the affinity. This mutant was named MDHPS. The amino acid sites corresponding to the receptor protein DHPS are (ARG238>TRP, GLU245>CYS, HIS284>TRP, SER235>VAL, VAL251>ILE).

[0054] Example 2: Preparation of the mutant MDHPS

[0055] This embodiment provides the preparation of the mutant MDHPS, and the specific steps are as follows.

[0056] A. Design of the full mdhps gene of mutant Streptococcus pneumoniae R6: Based on the MDHPS amino acid sequence obtained in Example 1, the encoded gene sequence SEQ ID NO.1 was obtained.

[0057] B. Using DHPS as the research object, the double-stranded gene to be synthesized was divided into 22 segments. Primers were designed, and restriction enzyme sites were inserted into segments 1 and 22. Specifically, an NheI restriction site was inserted in SEQ ID NO.3, and an XhoI restriction site was inserted in SEQ ID NO.24, so that there was a 15-16 bp overlap between each pair of adjacent segments. The 22 segments were spliced ​​together using overlap extension PCR technology, and the target fragment mdhps was obtained after splicing (Table 3).

[0058] Table 3 Primer design for mutant mdhps

[0059]

[0060]

[0061] C. Construction of the recombinant expression vector strain: The expression vector pET-28a was digested with NdeI and XhoI. The target fragment was ligated into the expression vector to obtain the recombinant expression vector pET-28a-mdhps. The recombinant expression vector pET-28a-mdhps was transformed into Escherichia coli BL21(DE3), and after screening, a recombinant expression strain that efficiently expressed recombinant Streptococcus pneumoniae dihydrofolate reductase was obtained. Figure 6 ).

[0062] D. Induced expression of recombinant MDHPS: In the OD of the expression strain culture system 600 When the concentration is 0.6-0.8, IPTG is added to the fermentation system to make the concentration of IPTG in the culture system 1mM.

[0063] E. Purification of recombinant MDHPS: The supernatant was subjected to nickel column affinity chromatography, and the eluent was collected. The elution buffer used in the nickel column affinity chromatography was prepared as follows: 20 mmol Tris, 0.5 mol NaCl, and 100 mmol imidazole were dissolved in water, the pH was adjusted to 7.9 with HCl, and then the volume was brought up to 1 L with water to obtain 1 L of elution buffer. The purified MDHPS were identified by SDS-PAGE, and the results are shown in the figure. Figure 7 .

[0064] The method for preparing recombinant DHPS, wherein step C specifically includes the following steps: picking single-clone recombinant expression strains to a concentration of 30 μg / mL -1 After overnight incubation at 37°C and 200 rpm in kanamycin culture medium, the overnight bacterial culture was diluted 100-fold with liquid culture medium and incubated at 37°C and 200 rpm for 3 hours. IPTG was added to the culture medium to a final concentration of 1M, and induction was performed at 16-28°C for 5-16 hours. The culture medium was centrifuged at 4°C, and the bacterial cell pellet was collected. The pellet was resuspended in resuspension buffer, sonicated, and the suspension was centrifuged, and the supernatant was collected.

[0065] Example 3 Thermal stability test of mutant MDHPS

[0066] Wild-type DHPS and mutant MDHPS were used as research subjects. The proteins were heat-treated at 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 30 min, and their binding capacity to the drug was measured. The maximum absorbance without heat treatment was plotted as 100%, and the percentage of the maximum absorbance at different temperatures to the maximum absorbance without heat treatment was calculated to determine the temperature at which the protein loses 50% of its binding capacity, i.e., the half-inactivation temperature (T0). 50 Both the wild-type and mutant strains were essentially inactivated after heat treatment at 70°C for 30 minutes. (For example...) Figure 8As shown, wild-type T 50 The value was 52.3℃, and the mutant's T... 50 The value was 55.5℃, which is 3.2℃ higher than that of the wild type, indicating improved thermal stability. This confirms that the method can be used to improve the thermal stability of proteins.

[0067] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> China Agricultural University <120> A method for preparing a thermostable dihydropteroate synthase mutant <130> KHP211124445.4 <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 942 <212> DNA <213> Artificial Sequence <400> 1 atgagcagca aagcgaacca tgcaaaaacc gtaatttgtg gtattattaa cgtgacgccg 60 gatagcttta gcgatggtgg tcagtttttt gcactggaac aggcgctgca gcaggcacgt 120 aaactgatcg cagaaggtgc atccatgctg gatattggtg gtgaaagcac acgccctggt 180 tcaagctatg ttgaaattga agaagaaatc cagcgtgttg ttccggttat taaagcaatt 240 cgtaaagaaa gcgatgttct gatttctatt gatacatgga aatcacaggt tgcagaagca 300 gcactggcgg caggtgcaga tctggttaat gatattaccg gtctgatggg tgatgaaaaa 360 atgccgcatg ttgttgcaga agcacgtgca caggttgtta ttatgtttaa tccggttatg 420 gcacgtccgc agcatccgag cagcctgatt tttccgcatt ttggttttgg tcaggcattt 480 accgaagaag aactggcaga ttttgaaacc ctgccgattg aagaactgat ggaagcattt 540 tttgaacgtg cactggcacg tgcagcagaa gcaggtattg caccggaaaa tattctgctg 600 gatccgggta ttggttttgg tctgaccaaa aaagaaaatc tgctgctgct gcgtgatctg 660 gataaactgc atcagaaagg ttatccgatt tttctgggtg ttgttcgtaa atggtttgtt 720 attaatattc tgtgtgaaaa tggttttgaa attaatccgg aaaccgaact gggttttcgt 780 aatcgtgata ccgcaagcgc acatgttacc agcattgcag cacgtcaggg tgttgaagtt 840 gttcgtgttt gggatgttgc aagccatcgt atggcagttg aaattgcaag cgcaattcgt 900 ctggcagatg aagcagaaaa tctggatctg aaacagtata aa 942 <210> 2 <211> 314 <212> PRT <213> Artificial Sequence <400> 2 Met Ser Ser Lys Ala Asn His Ala Lys Thr Val Ile Cys Gly Ile Ile 1 5 10 15 Asn Val Thr Pro Asp Ser Phe Ser Asp Gly Gly Gln Phe Phe Ala Leu 20 25 30 Glu Gln Ala Leu Gln Gln Ala Arg Lys Leu Ile Ala Glu Gly Ala Ser 35 40 45 Met Leu Asp Ile Gly Gly Glu Ser Thr Arg Pro Gly Ser Ser Tyr Val 50 55 60 Glu Ile Glu Glu Glu Ile Gln Arg Val Val Pro Val Ile Lys Ala Ile 65 70 75 80 Arg Lys Glu Ser Asp Val Leu Ile Ser Ile Asp Thr Trp Lys Ser Gln 85 90 95 Val Ala Glu Ala Ala Leu Ala Ala Gly Ala Asp Leu Val Asn Asp Ile 100 105 110 Thr Gly Leu Met Gly Asp Glu Lys Met Pro His Val Val Ala Glu Ala 115 120 125 Arg Ala Gln Val Val Ile Met Phe Asn Pro Val Met Ala Arg Pro Gln 130 135 140 His Pro Ser Ser Leu Ile Phe Pro His Phe Gly Phe Gly Gln Ala Phe 145 150 155 160 Thr Glu Glu Glu Leu Ala Asp Phe Glu Thr Leu Pro Ile Glu Glu Leu 165 170 175 Met Glu Ala Phe Phe Glu Arg Ala Leu Ala Arg Ala Ala Glu Ala Gly 180 185 190 Ile Ala Pro Glu Asn Ile Leu Leu Asp Pro Gly Ile Gly Phe Gly Leu 195 200 205 Thr Lys Lys Glu Asn Leu Leu Leu Leu Arg Asp Leu Asp Lys Leu His 210 215 220 Gln Lys Gly Tyr Pro Ile Phe Leu Gly Val Val Arg Lys Trp Phe Val 225 230 235 240 Ile Asn Ile Leu Cys Glu Asn Gly Phe Glu Ile Asn Pro Glu Thr Glu 245 250 255 Leu Gly Phe Arg Asn Arg Asp Thr Ala Ser Ala His Val Thr Ser Ile 260 265 270 Ala Ala Arg Gln Gly Val Glu Val Val Arg Val Trp Asp Val Ala Ser 275 280 285 His Arg Met Ala Val Glu Ile Ala Ser Ala Ile Arg Leu Ala Asp Glu 290 295 300 Ala Glu Asn Leu Asp Leu Lys Gln Tyr Lys 305 310 <210> 3 <211> 56 <212> DNA <213> Artificial Sequence <400> 3 cagcggcctg gtgccgcgcg gcagccatat gagcagcaaa gcgaaccatg caaaaa 56 <210> 4 <211> 62 <212> DNA <213> Artificial Sequence <400> 4 atcgctaaag ctatccggcg tcacgttaat aatacccacaa attacggttt ttgcatggtt 60 cg 62 <210> 5 <211> 62 <212> DNA <213> Artificial Sequence <400> 5 gatagcttta gcgatggtgg tcagtttttt gcactggaac aggcgctgca gcaggcacgt 60 aa 62 <210> 6 <211> 62 <212> DNA <213> Artificial Sequence <400> 6 tgctttcacc accaatatcc agcatggatg caccttctgc gatcagttta cgtgcctgct 60 gc 62 <210> 7 <211> 62 <212> DNA <213> Artificial Sequence <400> 7 ttggtggtga aagcacacgc cctggttcaa gctatgttga aattgaagaa gaaatccagc 60 gt 62 <210> 8 <211> 61 <212> DNA <213> Artificial Sequence <400> 8 tcagaacatc gctttcttta cgaattgctt taataaccgg aacaacacgc tggatttctt 60 c 61 <210> 9 <211> 64 <212> DNA <213> Artificial Sequence <400> 9 aaagcgatgt tctgatttct attgatacat ggaaatcaca ggttgcagaa gcagcactgg 60 cggc 64 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <400> 10 tcatcaccca tcagaccggt aatatcatta accagatctg cacctgccgc cagtgctgct 60 <210> 11 <211> 62 <212> DNA <213> Artificial Sequence <400> 关于专利文本的翻译,我已尽力按照要求进行翻译。请注意,由于原始文本中包含一些特定的专利标识和格式,翻译后的文本可能在格式上与原始文本不完全一致,但我已尽力保留了所有相关信息。如果您对翻译结果有任何疑问或需要进一步的帮助,请随时告诉我。11 tctgatgggt gatgaaaaaa tgccgcatgt tgttgcagaa gcacgtgcac aggttgttat 60 ta 62 <210> 12 请注意,专利文本的翻译需要非常准确和专业,以确保专利的有效性和可理解性。如果可能的话,建议咨询专业的专利翻译人员或律师进行审核。上述翻译仅供参考。 <211> 62 <212> DNA <213> Artificial Sequence <400> 12 aatcaggctg ctcggatgct gcggacgtgc cataaccgga ttaaacataa taacaacctg 60 tg 62 <210> 13 <211> 62 <212> DNA <213> Artificial Sequence <400> 13 ccgagcagcc tgatttttcc gcattttggt tttggtcagg catttaccga agaagaactg 60 gc 62 <210> 14 <211> 62 <212> DNA <213> Artificial Sequence <400> 14 caaaaaatgc ttccatcagt tcttcaatcg gcagggtttc aaaatctgcc agttcttctt 60 cg 62 <210> 15 <211> 62 <212> DNA <213> Artificial Sequence <400> 15 tggaagcattttttgaacgt gcactggcac gtgcagcaga agcaggtatt gcaccggaaa 60 at 62 <210> 16 <211> 62 <212> DNA <213> Artificial Sequence <400> 16 ttttcttttt tggtcagacc aaaaccaata cccggatcca gcagaatatt ttccggtgca 60 at 62 <210> 17 <211> 62 <212> DNA <213> Artificial Sequence <400> 17 gaccaaaaaa gaaaatctgc tgctgctgcg tgatctggat aaactgcatc agaaaggtta 60 tc 62 <210> 18 <211> 56 <212> DNA <213> Artificial Sequence <400> 18 attaataaca aaccatttac gaacaacacc cagaaaaatc ggataacctt tctgat 56 <210> 19 <211> 56 <212> DNA <213> Artificial Sequence <400> 19 tggtttgtta ttaatattct gtgtgaaaat ggttttgaaa ttaatccgga aaccga 56 <210> 20 <211> 62 <212> DNA <213> Artificial Sequence <400> 20 tgctggtaac atgtgcgctt gcggtatcac gattacgaaa acccagttcg gtttccggat 60 ta 62 <210> twenty one <211> 62 <212> DNA <213> Artificial Sequence <400> twenty one cacatgttac cagcattgca gcacgtcagg gtgttgaagt tgttcgtgtt tgggatgttg 60 ca 62 <210> twenty two <211> 62 <212> DNA <213> Artificial Sequence <400> twenty two tctgccagac gaattgcgct tgcaatttca actgccatac gatggcttgc aacatcccaa 60 ac 62 <210> twenty three <211> 61 <212> DNA <213> Artificial Sequence <400> twenty three caattcgtct ggcagatgaa gcagaaaatc tggatctgaa acagtataaa taactcgagc 60 a 61 <210> twenty four <211> 57 <212> DNA <213> Artificial Sequence <400> twenty four gctttgttag cagccggatc tcagtggtgg tggtggtggt gctcgagtta tttatac 57

Claims

1. A dihydropteroate synthase mutant MDHPS, characterized in that, The amino acid sequence of the dihydropteroate synthase mutant MDHPS is shown in SEQ ID NO.

2.

2. The encoding gene of the dihydropteroate synthase mutant MDHPS according to claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. A biomaterial, characterized in that, The biological material can express the dihydropteroate synthase mutant MDHPS as described in claim 1, or the biological material contains the coding gene as described in claim 2; the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector, engineered bacteria, or transgenic cell line.

4. The application of the dihydropteroate synthase mutant MDHPS of claim 1, the encoding gene of claim 2, or the biomaterial of claim 3 in the detection of sulfonamide drug residues.

5. A reagent or kit for detecting sulfonamide drug residues, characterized in that, The reagent or kit uses the dihydropteroate synthase mutant MDHPS as described in claim 1 as the receptor protein for the detection of sulfonamide drug residues.

6. A method for detecting sulfonamide drug residues, characterized in that, The dihydropteroate synthase mutant MDHPS described in claim 1 was used as the immune receptor protein for the detection of sulfonamide drug residues.

7. A method for preparing a thermostable receptor protein for detecting sulfonamide drug residues, characterized in that, The heat-stable receptor protein is the dihydropteroate synthase mutant MDHPS as described in claim 1; The specific steps include: encoding the target fragment of the dihydropteroate synthase mutant MDHPS. mdhps The nucleotide sequence shown in SEQ ID NO. 3-24 was obtained by splicing together using overlap extension PCR technology, with an insertion into the nucleotide sequence shown in SEQ ID NO.

3. NheI The restriction enzyme site is inserted into the nucleotide sequence shown in SEQ ID NO.

24. XhoI Enzyme cleavage sites; The expression vector pET-28a was double-digested with NdeI and XhoI, and the target fragment was ligated into the expression vector to obtain the recombinant expression vector pET-28a-mdhps. The recombinant expression vector pET-28a-mdhps was transformed into Escherichia coli to obtain a recombinant expression strain that efficiently expresses recombinant Streptococcus pneumoniae dihydrofolate reductase. When the OD600 of the expression strain culture system is 0.6-0.8, IPTG is added to the expression strain culture system, and the mixture is induced at 16-28℃ for 5-16 h to obtain the dihydropteroate synthase mutant MDHPS.

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