A low immunogenic asparaginase mutant and its preparation method and application

Abstract

The present invention provides a low-immunogenicity asparaginase mutant, in which T119 of wild-type asparaginase type II is replaced by C, K196 is replaced by A, Y218 is replaced by D or C or E, Y220 is replaced by A, and Y289 is replaced by R or G or K; and a preparation method and application thereof are provided. Compared with the prior art, the present invention has the following advantages: the low-immunogenicity asparaginase mutant provided by the present invention, its preparation method and application, comprehensively considered, the asparaginase mutant prepared by this preparation method has high yield, high purity, high safety and low cost, and at the same time, its immunogenicity is effectively reduced by mutation, avoiding progressive immune response and systemic allergic reaction.

Description

Technical Field

[0001] The present invention relates to the technical field of gene recombination expression, and in particular to a low-immunogenicity asparaginase mutant and a preparation method and application thereof. Technical Background

[0002] As one of the important drugs in the treatment of acute lymphoblastic leukemia (ALL) in children and adults, researchers have been exploring it for a long time. Between 1958 and 1962, Broome confirmed that the active substance in guinea pig serum that inhibits tumor growth is L-Asp. Subsequently, Mashburn et al. discovered that L-Asp can be extracted from Escherichia coli, which is also the most commonly used L-Asp preparation today. Since the extraction process of this type of drug was formed in the 1960s and 1970s, the protein purification technology was relatively primitive, the impurity content in the product was high, and asparaginase is a foreign protein to the human body, it is more likely to cause allergic reactions when used.

[0003] Some tumor cells (such as lymphoid leukemia cells) lack asparaginase and cannot synthesize asparagine, and must rely on the host to supply it. After administration of asparaginase, asparagine in the extracellular fluid is hydrolyzed into aspartic acid, which causes tumor cells to lack asparagine, affecting protein synthesis, inhibiting tumor cell growth, and eventually leading to death. Normal cells are spared because they have the ability to synthesize asparagine. The hydrolysis reaction of asparaginase is as follows: Figure 1 shown.

[0004] Proteins with L-asparagine amidohydrolase activity, commonly known as L-asparaginase, have been successfully used to treat various potentially fatal diseases, including acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL), with children constituting a large proportion of patients suffering from these diseases.

[0005] However, existing microbial-derived L-asparaginase often causes progressive immune reactions and systemic allergic reactions in clinical practice, which limits its clinical application. How to obtain L-asparaginase with low immunogenicity has become a research focus. There are currently two reasons for the high immunogenicity of L-asparaginase: first, microbial-derived L-asparaginase is a heterologous protein to the human body and is easy to cause an immune response in the human body; second, L-asparaginase derived from Escherichia coli, the obtained product is mixed with L-asparaginase expressed by Escherichia coli background, the product purity is uneven, and there is a potential immunogenicity safety risk. Summary of the invention

[0006] In view of the above-mentioned limitations, the present invention proposes a low-immunogenicity asparaginase mutant and a preparation method and application thereof, which overcome the deficiencies and defects mentioned in the background technology.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A low immunogenic asparaginase mutant, wherein T119 of wild-type asparaginase type II is replaced by C, K196 is replaced by A, Y218 is replaced by D, C or E, Y220 is replaced by A, and Y289 is replaced by R, G or K.

[0009] Escherichia coli produces two types of asparaginase, namely L-asparaginase type I and L-asparaginase type II. In the present invention, wild-type asparaginase type II (GenBank: WP_000394140.1) is used as a basis for mutation optimization. The optimized mutant asparaginase consists of 326 amino acids, as shown in SEQ ID NO.1-9, and its gene sequence is shown in SEQ ID NO.10-18, wherein all sequence mutation sites are shown in Table 1 below. The amino acid sequences shown in SEQ ID NO.1-9 are named low immunogenic asparaginase mutants Etb-1, Etb-2, Etb-3, Etb-4, Etb-5, Etb-6, Etb-7, Etb-8, and Etb-9, respectively.

[0010] Table 1

[0011] no. T119 K196 Y218 Y220 Y289 mut1 C A D A R mut2 C A D A G mut3 C A D A K mut4 C A C A R mut5 C A C A G mut6 C A C A K mut7 C A E A R mut8 C A E A G mut9 C A E A K

[0012] It can be seen from Table 1 that the mutant asparaginase is based on the 326-position amino acid sequence of the wild-type asparaginase type II, with a mutation from T to C at position 119, a mutation from K to A at position 196, a mutation from Y to D or C or E at position 218, a mutation from Y to A at position 220, and a mutation from Y to R or G or K at position 289.

[0013] The amino acid sequence at position 326 of wild-type asparaginase type II is as follows:

[0014] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPS T SMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPAR K HTSDTPFDVSKLNELPKVGIV Y N YANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAK Y GFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY.

[0015] Optionally, the above-mentioned low immunogenic asparaginase mutant, the amino acid sequence of the asparaginase mutant is shown in any one of SEQ ID No.1 to SEQ ID No.9.

[0016] Amino acid sequence SEQ ID NO.1 (T119C K196A Y218D Y220A Y289R):

[0017] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKT NTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVDNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKRGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0018] Amino acid sequence SEQ ID NO.2 (T119C K196A Y218D Y220A Y289G):

[0019] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVCNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKRGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0020] Amino acid sequence SEQ ID NO.3 (T119C K196A Y218D Y220A Y289K):

[0021] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVENAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKRGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0022] Amino acid sequence SEQ ID NO.4 (T119C K196A Y218C Y220A Y289R):

[0023] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVDNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKGGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0024] Amino acid sequence SEQ ID NO.5 (T119C K196A Y218C Y220A Y289G):

[0025] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVDNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKKGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0026] Amino acid sequence SEQ ID NO.6 (T119C K196A Y218C Y220A Y289K):

[0027] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVCNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKGGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0028] Amino acid sequence SEQ ID NO.7 (T119C K196A Y218E Y220A Y289R):

[0029] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVCNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKKGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0030] Amino acid sequence SEQ ID NO.8 (T119C K196A Y218E Y220A Y289G):

[0031] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKT NTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVENAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKGGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0032] Amino acid sequence SEQ ID NO.9 (T119C K196A Y218E Y220A Y289K):

[0033] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKT NTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVENAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKKGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY.

[0034] Optionally, in the above-mentioned low immunogenic asparaginase mutant, the sequence encoding the amino acid is a polynucleotide sequence, and the polynucleotide sequence is shown in any one of SEQ ID NO.10 to SEQ ID NO.18.

[0035] Polynucleotide sequence SEQ ID NO.10:

[0036] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgataacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaagatggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0037] Polynucleotide sequence SEQ ID NO.11:

[0038] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgataacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaggcggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0039] Polynucleotide sequence SEQ ID NO.12:

[0040] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgataacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaaaaggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0041] Polynucleotide sequence SEQ ID NO.13:

[0042] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgtttgcaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaacgcggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0043] Polynucleotide sequence SEQ ID NO.14:

[0044] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgtttgcaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaggcggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0045] Polynucleotide sequence SEQ ID NO.15:

[0046] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgtttgcaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaaaaggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0047] Polynucleotide sequence SEQ ID NO.16:

[0048] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgaaaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaagaggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0049] Polynucleotide sequence SEQ ID NO.17:

[0050] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgaaaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaggcggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0051] Polynucleotide sequence SEQ ID NO.18:

[0052] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgaaaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaaaaggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0053] The second object of the present invention is to provide a recombinant expression vector comprising the above-mentioned polynucleotide sequence encoding the amino acid sequence of the low immunogenic asparaginase mutant.

[0054] The third object of the present invention is to provide a recombinant bacterium, comprising the above-mentioned recombinant expression vector, wherein the recombinant bacterium is obtained by transforming the strain with the recombinant expression vector.

[0055] Optionally, the above-mentioned recombinant bacteria is a strain of Escherichia coli; preferably, it is a strain of Escherichia coli TOP10.

[0056] The fourth object of the present invention is to provide a method for preparing the above-mentioned low immunogenic asparaginase mutant, comprising the following steps:

[0057] S1. Preparation of low immunogenic asparaginase mutant expression strain:

[0058] Using any one of the polynucleotide sequences SEQ ID NO.10 to SEQ ID NO.18 synthesized by the whole gene as a template, using primers to perform PCR amplification, purifying the PCR product, double-digesting it and inserting it into a plasmid, transforming Escherichia coli competent cells, selecting positive clones, extracting the plasmid and verifying it by sequencing, transforming the plasmid into competent cells, selecting positive clones, and obtaining an expression strain of a low-immunogenic asparaginase mutant;

[0059] S2. Expression of low immunogenic asparaginase:

[0060] The expression strain obtained in step S1 is fermented on LB medium and gradually expanded, and the bacterial cells are collected by centrifugation;

[0061] S3. Purification of low immunogenic asparaginase:

[0062] The sludge-like bacterial cells obtained in step S2 are disrupted with a lysis solution, and the supernatant is collected by centrifugation. The supernatant is eluted through a chromatography column, and the eluate is concentrated to obtain purified low-immunogenic asparaginase.

[0063] The fifth object of the present invention is to provide the use of the above-mentioned low-immunogenicity asparaginase mutant, the above-mentioned recombinant expression vector, the above-mentioned recombinant bacteria and the above-mentioned purified low-immunogenicity asparaginase in the preparation of a preparation for treating lymphatic system malignancies;

[0064] The malignant tumor of the lymphatic system is preferably acute lymphoblastic leukemia ALL or lymphocytic lymphoma LBL;

[0065] More preferably, it is acute lymphoblastic leukemia ALL.

[0066] Optionally, in the above application, the preparation is a lyophilized preparation with an asparaginase content of 20 g / L.

[0067] Optionally, in the above application, the preparation further contains 0.38 g / L-1.02 g / L of anhydrous disodium hydrogen phosphate, 0.39 g / L-1.01 g / L of sodium dihydrogen phosphate monohydrate, 4.8 g / L of sodium chloride, 50 ppm of Tween 80 and 30 g / L-50 g / L of mannitol, and the pH value of the preparation is 6.5-7.2.

[0068] Compared with the prior art, the present invention has the following advantages:

[0069] The low immunogenicity asparaginase mutant provided by the present invention and its preparation method and application use advanced protein preparation technology, so that the enzyme purity is higher, the host bacterial protein residue and DNA residue meet or exceed the current pharmacopoeia standard requirements, and the safety is greatly improved. At the same time, the expression system and gene provided by the present invention are optimized to make the asparaginase yield per unit volume higher and the drug cost lower; comprehensively considered, the asparaginase mutant prepared by this preparation method has high yield and high purity, and its immunogenicity is effectively reduced by mutation, avoiding progressive immune response and systemic allergic reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Shown is the hydrolysis reaction formula of asparaginase.

[0071] Figure 2 Shown is a plasmid map of pET42a-etb-1.

[0072] Figure 3 The plasmid maps are shown as pET42a-etb-2, pET42a-etb-3, pET42a-etb-4, pET42a-etb-5, pET42a-etb-6, pET42a-etb-7, pET42a-etb-8, and pET42a-etb-9. Figure 3 AH show the plasmid maps of pET42a-etb-2 to pET42a-etb-9, respectively.

[0073] Figure 4 Shown is the NdeⅠ and XhoⅠ restriction map of pET42a-etb-1 plasmid.

[0074] Figure 5 It shows the NdeⅠ and XhoⅠ restriction enzyme digestion map of pET42a-etb-2 / 3 / 6 / 8 plasmid; in the figure, channel 1 is the marker, and channels 3 to 6 are the double enzyme digestion verification results of pET42A-etb-2 / 3 / 6 / 8.

[0075] Figure 6 Shown is the SDS-PAGE electrophoresis pattern of purified low-immunogenic asparaginase; well 7 in the figure is a marker, and wells 1-6 are the electrophoresis patterns of pET42a-etb-1 / 2 / 4 / 5 / 8 / 9 proteins, respectively.

[0076] Figure 7 The graph shows the dose-effect relationship of drug action time at different cell inoculation amounts when 20,000 cells / well are used.

[0077] Figure 8 The graph shows the dose-effect relationship of drug action time at different cell inoculation amounts at 25,000 cells / well.

[0078] Fig. 9 The graph shows the dose-effect relationship of drug action time at different cell inoculation amounts when 30,000 cells / well are used.

[0079] Fig.10 Shown are the results of the inhibitory activity analysis of low-immunogenic asparaginase against Nalm-6.

[0080] Fig.11 Shown are specific results of the screening threshold value (SCP) and the titer threshold value (TCP) in one embodiment of the present invention.

[0081] Fig.12 It shows the specific result of threshold CCP calculation in one embodiment of the present invention. DETAILED DESCRIPTION

[0082] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not used to limit the scope of the present invention.

[0083] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0084] In order to further understand the present invention, the present invention is further described in detail below in conjunction with the best embodiment.

[0085] Example 1

[0086] A low immunogenic asparaginase mutant, wherein T119 of wild-type asparaginase type II is replaced by C, K196 is replaced by A, Y218 is replaced by D, C or E, Y220 is replaced by A, and Y289 is replaced by R, G or K.

[0087] Escherichia coli produces two types of asparaginase, namely L-asparaginase type I and L-asparaginase type II. In the present invention, wild-type asparaginase type II (GenBank: WP_000394140.1) is used as a basis for mutation optimization. The optimized mutant asparaginase consists of 326 amino acids, as shown in SEQ ID NO.1-9, and its gene sequence is shown in SEQ ID NO.10-18, wherein all sequence mutation sites are shown in Table 1. The amino acid sequences shown in SEQ ID NO.1-9 are named low immunogenic asparaginase mutants Etb-1, Etb-2, Etb-3, Etb-4, Etb-5, Etb-6, Etb-7, Etb-8, and Etb-9, respectively.

[0088] It can be seen from Table 1 that the mutant asparaginase is based on the 326-position amino acid sequence of the wild-type asparaginase type II, with a mutation from T to C at position 119, a mutation from K to A at position 196, a mutation from Y to D or C or E at position 218, a mutation from Y to A at position 220, and a mutation from Y to R or G or K at position 289.

[0089] The amino acid sequence at position 326 of wild-type asparaginase type II is as follows:

[0090] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPS T SMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPAR K HTSDTPFDVSKLNELPKVGIV Y N Y ANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAK Y GFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY.

[0091] The amino acid sequences of the asparaginase mutants are shown as any one of SEQ ID No.1 to SEQ ID No.9.

[0092] Amino acid sequence SEQ ID NO.1 (T119C K196A Y218D Y220A Y289R):

[0093] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVDNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKRGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0094] Amino acid sequence SEQ ID NO.2 (T119C K196A Y218D Y220A Y289G):

[0095] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVCNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKRGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0096] Amino acid sequence SEQ ID NO.3 (T119C K196A Y218D Y220A Y289K):

[0097] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVENAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKRGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0098] Amino acid sequence SEQ ID NO.4 (T119C K196A Y218C Y220A Y289R):

[0099] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVDNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKGGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0100] Amino acid sequence SEQ ID NO.5 (T119C K196A Y218C Y220A Y289G):

[0101] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVDNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKKGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0102] Amino acid sequence SEQ ID NO.6 (T119C K196A Y218C Y220A Y289K):

[0103] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVCNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKGGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0104] Amino acid sequence SEQ ID NO.7 (T119C K196A Y218E Y220A Y289R):

[0105] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVCNAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKKGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0106] Amino acid sequence SEQ ID NO.8 (T119C K196A Y218E Y220A Y289G):

[0107] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVENAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKGGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY;

[0108] Amino acid sequence SEQ ID NO.9 (T119C K196A Y218E Y220A Y289K):

[0109] LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSCSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKT NTTDVATFKSVNYGPLGYIHNGKIDYQRTPARAHTSDTPFDVSKLNELPKVGIVENAANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKKGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY.

[0110] The sequence encoding the amino acid is a polynucleotide sequence, and the polynucleotide sequence is shown in any one of SEQ ID NO.10 to SEQ ID NO.18.

[0111] Polynucleotide sequence SEQ ID NO.10:

[0112] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgataacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaagatggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0113] Polynucleotide sequence SEQ ID NO.11:

[0114] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgataacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaggcggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0115] Polynucleotide sequence SEQ ID NO.12:

[0116] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgataacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaaaaggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0117] Polynucleotide sequence SEQ ID NO.13:

[0118] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgtttgcaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaacgcggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0119] Polynucleotide sequence SEQ ID NO.14:

[0120] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgtttgcaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaggcggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0121] Polynucleotide sequence SEQ ID NO.15:

[0122] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgtttgcaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaaaaggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0123] Polynucleotide sequence SEQ ID NO.16:

[0124] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgaaaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaagaggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0125] Polynucleotide sequence SEQ ID NO.17:

[0126] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgaaaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaggcggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0127] Polynucleotide sequence SEQ ID NO.18:

[0128] ttacccaatatcaccattttagcaaccggcgggaccattgccggtggtggtgactccgcaaccaaatctaactacacagtgggtaaagttggcgtagaaaatctggttaatgcggtgccgcaactaaaagacattgcgaacgttaaaggcgagcaggtagtgaatatcggctcccaggacatgaacgataatgtctggctgacactggcgaaaaaaattaacaccgactgcgataagaccgacggcttcgtcattacccacggtaccgacacgatggaagaaactgcttacttcctcgacctgacggtgaaatgcgacaaaccggtggtgatggtcggcgcaatgcgtccgtcctgctctatgagcgcagacggtccattcaacctgtataacgcggtagtgaccgcagctgataaagcctccgccaaccgtggcgtgctggtagtgatgaatgacaccgtgcttgatggccgtgacgtcaccaaaaccaacaccaccgacgtagcgaccttcaagtctgttaactacggtcctctgggttacattcacaacggtaagattgactaccagcgtaccccggcacgtgcgcataccagcgacacgccattcgatgtctctaagctgaatgaactgccgaaagtcggcattgttgaaaacgcggctaacgcatccgatcttccggctaaagcactggtagatgcgggctatgatggcatcgttagcgctggtgtgggtaacggcaacctgtataaatctgtgttcgacacgctggcgaccgccgcgaaaaccggtactgcagtcgtgcgttcttcccgcgtaccgacgggcgctaccactcaggatgccgaagtggatgatgcgaaaaaaggcttcgtcgcctctggcacgctgaacccgcaaaaagcgcgcgttctgctgcaactggctctgacgcaaaccaaagatccgcagcagatccagcagatcttcaatcagtactaa。

[0129] Example 2

[0130] A recombinant expression vector comprises the polynucleotide sequence SEQ ID NO.10-SEQ ID NO.18 encoding the amino acid sequence of the low immunogenic asparaginase mutant.

[0131] A recombinant bacterium comprises the above-mentioned recombinant expression vector, wherein the recombinant bacterium is obtained by transforming the strain with the recombinant expression vector.

[0132] The recombinant bacterial strain is Escherichia coli, preferably the Escherichia coli BL21 (DE3) strain.

[0133] Example 3

[0134] A method for preparing a low-immunogenic asparaginase mutant comprises the following steps:

[0135] S1. Preparation of low immunogenic asparaginase mutant expression strain:

[0136] Using any one of the polynucleotide sequences SEQ ID NO.10 to SEQ ID NO.18 synthesized by the whole gene as a template, using primers to perform PCR amplification, purifying the PCR product, double-digesting it and inserting it into a plasmid, transforming Escherichia coli competent cells, selecting positive clones, extracting the plasmid and verifying it by sequencing, transforming the plasmid into competent cells, selecting positive clones, and obtaining an expression strain of a low-immunogenic asparaginase mutant;

[0137] S2. Expression of low immunogenic asparaginase:

[0138] The expression strain obtained in step S1 is fermented on LB medium and gradually expanded, and the bacterial cells are collected by centrifugation;

[0139] S3. Purification of low immunogenic asparaginase:

[0140] The sludge-like bacterial cells obtained in step S2 are disrupted with a lysis solution, and the supernatant is collected by centrifugation. The supernatant is eluted through a chromatography column, and the eluate is concentrated to obtain purified low-immunogenic asparaginase.

[0141] Example 4

[0142] Preparation, expression and purification of low immunogenic asparaginase mutant Etb-1:

[0143] 1. Preparation of low immunogenic asparaginase mutant Etb-1:

[0144] The polynucleotide sequence gene shown in SEQ ID NO.10 synthesized by the whole gene was used as a template, pFetb-1 / pRetb-1, and pFetb-1y / pRetb-1 were used as upstream and downstream primers, and the gene was amplified by two-step PCR to introduce NdeⅠ / XhoⅠ restriction sites and signal peptide sequences. The first step of PCR: using the synthetic gene as a template, using the high-fidelity DNA polymerase Phusion of Thermo Scientific, PCR was performed at 98℃ for 10 seconds, 55℃ for 30 seconds, 72℃ for 60 seconds, 30 cycles; 72℃ for 10 minutes. The obtained PCR product was recovered from the gel and used as a PCR template for the second step of PCR: using the high-fidelity DNA polymerase Phusion of Thermo Scientific, PCR was performed at 98℃ for 10 seconds, 55℃ for 30 seconds, 72℃ for 70 seconds, 30 cycles; 72℃ for 10 minutes. The obtained PCR product was recovered from the gel (about 1050 bp in size), and then double digested with XhoI / NdeI and inserted into the plasmid pET42a (Invitrogen product). The competent E. coli Top 10 was transformed, and positive clones were selected on LB medium containing kanamycin. The plasmid was extracted and sequenced to verify the correctness. The plasmid was named pET42a-etb-1. The plasmid map is shown in Figure 2 As shown, the enzyme cleavage pattern is as Figure 4 As shown, pET42a-etb-1 was transformed into competent BL21 (DE3) (product of Thermo Scientific), selected on LB plates containing kanamycin sulfate (50 μg / ml), and the positive clones were preserved after verification.

[0145] The primer sequences are as follows (SEQ ID NO.19-SEQ ID NO.21):

[0146] pFetb-1 (SEQ ID NO.19):

[0147] CTGGTTATGGGTTTTAGTGGTGCAGCATTGGCATTACCCAATATCACCATTTTAG;

[0148] pFebt-1y (SEQ ID NO.20):

[0149] CTACATATGGAGTTTTTCAAAAAGACGGCACTTGCCGCACTGGTTATGGGTTTTAGTG;

[0150] pRetb-1 (SEQ ID NO.21):

[0151] GTGCTCGAGTTAGTACTGATTGAAG.

[0152] 2. Low immunogenicity asparaginase expression:

[0153] Take the clone prepared above, inoculate it in 5mL LB medium containing kanamycin sulfate (50μg / ml), culture it in a shaker at 37℃ and 200RPM for about 3h, then transfer it to 100mL LB medium containing kanamycin sulfate (50μg / ml) and continue to culture it for about 3h as a seed solution for fermentation. The above seed solution is inoculated into a 5L fermenter (containing 2.5L fermentation basal medium), cultured at 30℃, and after the dissolved oxygen rises, the carbon source medium and the nitrogen source medium are added in batches, and the fermentation pH is controlled within the range of 7.0±0.1 with ammonia water. After the OD reaches 20-30, cool it to 28℃ and add IPTG (AMRESCO product) with a final concentration of 0.2mM, continue to culture for 5-6h, and collect the bacteria by centrifugation.

[0154] The culture medium formula is as follows:

[0155] LB liquid culture medium formula: peptone 10.0 g, yeast powder 5.0 g, sodium chloride 10.0 g, add purified water to 1.0 L, adjust the pH value to 7.2, divide into Erlenmeyer flasks, and sterilize at 121°C for 20 min.

[0156] LB solid medium: 10.0 g of peptone, 5.0 g of yeast powder, 10.0 g of sodium chloride, 15.0 g of agar powder, add purified water to 1.0 L, adjust the pH to 7.2, dispense into Erlenmeyer flasks, and sterilize at 121°C for 20 min.

[0157] Fermentation basal medium: same as LB medium, volume 2.5L, autoclave at 121℃ for 30min.

[0158] Feed nitrogen source medium: peptone 55g / L, yeast powder 35g / L, volume 0.5L, sterilization at 121℃ for 20min.

[0159] Feed carbon source culture medium: glucose 250 g / L, magnesium sulfate 5 g / L, volume 0.5 L, sterilized at 115°C for 20 min.

[0160] 3. Purification of low immunogenic asparaginase:

[0161] Take 10g of the bacterial mud collected in step 2, add it to 80mMTris lysis solution at 1:15 (g / g), and stir it with a tissue grinder to make it evenly dispersed. Use a high-pressure homogenizer at 700bar to break the bacteria, break it a total of 4 times, and collect the supernatant by centrifugation. The supernatant is loaded with the AKTA purifier 100 system, the chromatography column is the 25mm*300mm model chromatography column of Shanghai Xiamei Biochemical Technology Development Co., Ltd., and the filler is 100mL of QFF filler from GE Medical Devices. Rinse with a buffer containing 20mMTris 50mMNaCl pH9.30, and elute with a buffer containing 20mMTris 450mMNaCl pH9.30. The obtained eluate is concentrated and replaced with a medium-pure equilibrium solution (10mMTris pH7.60) using 10KD hollow fiber from Spectrum to obtain a medium-pure sample. The medium-pure sample was loaded on the AKTA purifier 100 system, the chromatography column was a 25mm*300mm chromatography column from Shanghai Xiamei Biochemical Technology Development Co., Ltd., and the filler was a 100mL QFF filler from GE Medical Devices. It was rinsed with 10mMTris50mMNaCl pH7.60 buffer, eluted with 10mMTris 150mMNaCl pH7.60, and the eluate was collected. The obtained eluate was concentrated and replaced with the 10KD hollow fiber of Spectrum Pure to a pure equilibrium solution (10mM citric acid-trisodium citrate pH5.1) to obtain a pure sample. The pure sample was loaded on the AKTA purifier 100 system, the chromatography column was a 25mm*300mm chromatography column from Shanghai Xiamei Biochemical Technology Development Co., Ltd., the filler was 60mL of SPFF filler from GE Medical Devices, and the eluent was 10mM citric acid-trisodium citrate 50mMNaCl pH 5.1. The eluent was collected and concentrated with a 10KD ultrafiltration tube to a 10mM PBS solution pH 7.00-7.40 and stored at -20°C.

[0162] The low immunogenic asparaginase expressed and purified by the above preparation method was verified by SDS-PAGE electrophoresis to have a purity of more than 95%. Figure 6 shown.

[0163] Example 5

[0164] Preparation, expression and purification of low immunogenic asparaginase mutants Etb-2 to Etb-9:

[0165] 1. Preparation of low immunogenic asparaginase mutants Etb-2 to Etb-9:

[0166] Using the fully synthesized SEQ ID NO.11 to SEQ ID NO.18 as templates, pFetb-1 / pRetb-1, and pFetb-1y / pRetb-1 as upstream and downstream primers, two-step PCR amplification of the gene was performed, and NdeⅠ / XhoⅠ restriction sites and signal peptide sequences were introduced. The first step of PCR: using the synthetic gene as a template, using the high-fidelity DNA polymerase Phusion from Thermo Scientific, PCR was performed at 98℃ for 10 seconds, 55℃ for 30 seconds, 72℃ for 60 seconds, 30 cycles; 72℃ for 10 minutes. The obtained PCR product was recovered from the gel and used as a PCR template for the second step of PCR: using the high-fidelity DNA polymerase Phusion from Thermo Scientific, PCR was performed at 98℃ for 10 seconds, 55℃ for 30 seconds, 72℃ for 70 seconds, 30 cycles; 72℃ for 10 minutes. After the obtained PCR product was recovered from the gel (size about 1050bp), it was double-digested with XhoI / NdeI and inserted into the plasmid pET42a (Invitrogen product), transformed into E. coli competent Top 10, and positive clones were selected on LB medium containing kanamycin. The plasmids were extracted and sequenced to verify the correctness. The obtained plasmids were pET42a-etb-2, pET42a-etb-3, pET42a-etb-4, pET42a-etb-5, pET42a-etb-6, pET42a-etb-7, pET42a-etb-8, and pET42a-etb-9. The total length of the plasmid is 6064bp. The plasmid map is shown in Figure 2 , Figure 3 As shown, the restriction enzyme digestion patterns using NdeⅠ and XhoⅠ are as follows Figure 4 , Figure 5 As shown. The above plasmids were transformed into competent E. coli BL21 (DE3) (product of Thermo Scientific). Positive clones were selected and stored on LB plates containing 50 μg / ml kanamycin sulfate, and the resulting expression strains were Etb-2, Etb-3, Etb-4, Etb-5, Etb-6, Etb-7, Etb-8, and Etb-9.

[0167] The primer sequences are as follows (SEQ ID NO.19-SEQ ID NO.21):

[0168] pFetb-1 (SEQ ID NO.19):

[0169] CTGGTTATGGGTTTTAGTGGTGCAGCATTGGCATTACCCAATATCACCATTTTAG;

[0170] pFebt-1y (SEQ ID NO.20):

[0171] CTACATATGGAGTTTTTCAAAAAGACGGCACTTGCCGCACTGGTTATGGGTTTTAGTG;

[0172] pRetb-1 (SEQ ID NO.21):

[0173] GTGCTCGAGTTAGTACTGATTGAAG.

[0174] 2. Low immunogenicity asparaginase expression:

[0175] Take the clone prepared above, inoculate it in 5mL LB medium containing kanamycin sulfate (50μg / ml), culture it in a shaker at 37℃ and 200RPM for about 3h, then transfer it to 100mL LB medium containing kanamycin sulfate (50μg / ml) and continue to culture it for about 3h as a seed solution for fermentation. The above seed solution is inoculated into a 5L fermenter (containing 2.5L fermentation basal medium), cultured at 30℃, and after the dissolved oxygen rises, the carbon source medium and the nitrogen source medium are added in batches, and the fermentation pH is controlled within the range of 7.0±0.1 with ammonia water. After the OD reaches 20-30, cool it to 28℃ and add IPTG (AMRESCO product) with a final concentration of 0.2mM, continue to culture for 5-6h, and collect the bacteria by centrifugation.

[0176] The culture medium formula is as follows:

[0177] LB liquid culture medium formula: peptone 10.0 g, yeast powder 5.0 g, sodium chloride 10.0 g, add purified water to 1.0 L, adjust the pH value to 7.2, divide into Erlenmeyer flasks, and sterilize at 121°C for 20 min.

[0178] LB solid medium: 10.0 g of peptone, 5.0 g of yeast powder, 10.0 g of sodium chloride, 15.0 g of agar powder, add purified water to 1.0 L, adjust the pH to 7.2, dispense into Erlenmeyer flasks, and sterilize at 121°C for 20 min.

[0179] Fermentation basal medium: same as LB medium, volume 2.5L, autoclave at 121℃ for 30min.

[0180] Feed nitrogen source medium: peptone 55g / L, yeast powder 35g / L, volume 0.5L, sterilization at 121℃ for 20min.

[0181] Feed carbon source culture medium: glucose 250 g / L, magnesium sulfate 5 g / L, volume 0.5 L, sterilized at 115°C for 20 min.

[0182] 3. Purification of low immunogenic asparaginase:

[0183] Take 10g of the bacterial mud collected in step 2, add it to 80mMTris lysis solution at 1:15 (g / g), and stir it with a tissue grinder to make it evenly dispersed. Use a high-pressure homogenizer at 700bar to break the bacteria, break it a total of 4 times, and collect the supernatant by centrifugation. The supernatant is loaded with the AKTA purifier 100 system, the chromatography column is the 25mm*300mm model chromatography column of Shanghai Xiamei Biochemical Technology Development Co., Ltd., and the filler is 100mL of QFF filler from GE Medical Devices. Rinse with a buffer containing 20mMTris 50mMNaCl pH9.30, and elute with a buffer containing 20mMTris 450mMNaCl pH9.30. The obtained eluate is concentrated and replaced with a medium-pure equilibrium solution (10mMTris pH7.60) using 10KD hollow fiber from Spectrum to obtain a medium-pure sample. The medium-pure sample was loaded on the AKTA purifier 100 system, the chromatography column was a 25mm*300mm chromatography column from Shanghai Xiamei Biochemical Technology Development Co., Ltd., and the filler was a 100mL QFF filler from GE Medical Devices. It was rinsed with 10mMTris50mMNaCl pH7.60 buffer, eluted with 10mMTris 150mMNaCl pH7.60, and the eluate was collected. The obtained eluate was concentrated and replaced with the 10KD hollow fiber of Spectrum Pure to a pure equilibrium solution (10mM citric acid-trisodium citrate pH5.1) to obtain a pure sample. The pure sample was loaded on the AKTA purifier 100 system, the chromatography column was a 25mm*300mm chromatography column from Shanghai Xiamei Biochemical Technology Development Co., Ltd., the filler was 60mL of SPFF filler from GE Medical Devices, and the eluent was 10mM citric acid-trisodium citrate 50mMNaCl pH 5.1. The eluent was collected and concentrated with a 10KD ultrafiltration tube to a 10mM PBS solution pH 7.00-7.40 and stored at -20°C.

[0184] The low immunogenic asparaginase expressed and purified by the above preparation method was verified by SDS-PAGE electrophoresis to have a purity of more than 95%. Figure 6 shown.

[0185] Example 6

[0186] Use of a low-immunogenicity asparaginase mutant, a recombinant expression vector comprising a polynucleotide sequence encoding the amino acid sequence of the asparaginase mutant, a recombinant bacterium comprising the recombinant expression vector, and the purified low-immunogenicity asparaginase prepared by the above preparation method in the preparation of a preparation for treating lymphatic system malignancies;

[0187] The malignant tumor of the lymphatic system is preferably acute lymphoblastic leukemia ALL or lymphocytic lymphoma LBL;

[0188] More preferably, it is acute lymphoblastic leukemia ALL.

[0189] The preparation may be a lyophilized preparation with an asparaginase content of 20 g / L.

[0190] The preparation also contains 0.38 g / L-1.02 g / L of anhydrous disodium hydrogen phosphate, 0.39 g / L-1.01 g / L of sodium dihydrogen phosphate monohydrate, 4.8 g / L of sodium chloride, 50 ppm of Tween 80 and 30 g / L-50 g / L of mannitol, and the pH value of the preparation is 6.5-7.2.

[0191] The preparation process of the preparation is:

[0192] The frozen sample of the purified low-immunogenic asparaginase mutant prepared in Example 4 or Example 5 was thawed at room temperature, mixed according to the ratio shown in Table 2, and freeze-dried again after the mixing was completed.

[0193] Table 2

[0194]

[0195] Example 7

[0196] 1. Low immunogenicity asparaginase in vitro cell experiment:

[0197] The prepared preparation was evaluated by in vitro cytological evaluation using the representative cell line of acute lymphoblastic leukemia Nalm-6. Nalm-6 cells were passaged in RPMI1640 culture medium, and Nalm-6 cells in the logarithmic growth phase were taken and prepared with RPMI 1640 culture medium to contain 2.0×10 5 , 2.5×10 5 and 3.0×10 5The cell suspension of 10 cells was inoculated in a 96-well plate, 100 μl per well. The test sample was diluted to 400 ug / ml using RPMI 1640 culture medium, and 4-fold serial dilutions were performed based on this, with a total of 8 dilutions. The prepared test sample was added to the above 96-well plate, and RPMI 1640 was used as a negative control. Two replicate wells were made for each dilution, 100 μl per well; cultured at 37°C, 5% carbon dioxide saturated humidity for 2, 3, and 4 days. 10 μl CCK-8 was added to each well, reacted for 2-4 hours, and the absorbance was measured at a wavelength of 450 nm with 630 nm as a reference.

[0198] Figure 7-Figure 9 The dose-effect relationship of drug action time with different cell inoculation amounts at 20,000 cells / well, 25,000 cells / well and 30,000 cells / well is shown respectively.

[0199] Recombinant asparaginase (BH-01) (ie, Etb-1) has a significant killing effect on human leukemia cells Nalm-6, with a significant dose-effect relationship when the cell dosage is 25,000 to 30,000 cells / well, the action time is 3 to 4 days, and the measurement time is 2 to 4 hours.

[0200] Fig.10 Analysis of the inhibitory activity of asparaginase against Nalm-6 was shown to be low immunogenic.

[0201] Low-immunogenic asparaginase has a significant killing effect on human leukemia cells Nalm-6, with a significant dose-effect relationship when the cell dosage is 25,000 to 30,000 cells / well, the action time is 3 to 4 days, and the measurement time is 2 to 4 hours.

[0202] 2. Animal experiments on asparaginase:

[0203] Animal experiments were conducted using the prepared preparations, using 6-9 week old SD rats (SPF grade, Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), males weighing 190-290 g, and females weighing 170-270 g. The animal weights were measured 5 days before grouping (D-5), and the rats were randomly divided into 8 groups according to gender, and the specific grouping is shown in Table 3.

[0204] Table 3

[0205]

[0206] The drug was administered by intravenous infusion once a day for 14 consecutive days. The first day of administration was defined as D1. The administration volume was 10 mL / kg. The changes in body weight and food intake were recorded. The results are shown in Table 4.

[0207] Table 4

[0208]

[0209] Example 8

[0210] Asparaginase immunogenicity assay:

[0211] 1. Asparaginase dosing regimen:

[0212] 1) Experimental groups:

[0213] The prepared preparation Etb-1 was used for animal experiments, and three low, medium and high dose groups (2000, 6000, 18000 IU / kg) were set up. Animal immunogenicity analysis experiments were performed using 6-9 week old SD rats (SPF grade, Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), with male body weight of 262-290g and female body weight of 196-235g. Five days before administration (D-5), the animals were randomly divided into 4 groups according to gender, including 5 males and 5 females in the vehicle control group, asparaginase low, medium and high dose groups. The drug was administered once a day for 14 consecutive days, with a dosing volume of 10 ml / kg and a dosing rate of 2 ml / kg / min. The day of administration was defined as D1. The specific grouping is shown in Table 5.

[0214] Table 5

[0215] male serial number female serial number Vehicle control group 5 1-5 5 6-10 Low dose group 5 11-15 5 16-20 Medium dose group 5 21-25 5 26-30 High dose group 5 31-35 5 36-40

[0216] 2) Blood sample collection:

[0217] Blood was collected from the jugular vein or orbital vein after isoflurane anesthesia before administration (D-5) of the four groups of animals. About 0.4 ml of blood was collected from the jugular vein before planned euthanasia on D15 for drug administration antibody detection. The sampling tube with separation gel + coagulant was used to separate serum within 1 hour after blood collection (room temperature, centrifugation at 1500g for 10 minutes). The collected serum samples were analyzed for immunogenicity using ELISA.

[0218] 2. Establishment of ELISA immunogenicity analysis method:

[0219] 1) Sample preparation:

[0220] ① Negative control sample (NC): 10 blank SD rat sera from different sources were mixed in equal volumes;

[0221] ② Screening and confirmation threshold samples: 30 blank SD rat sera were selected;

[0222] ③ Use negative control samples to dilute the positive antibody to 2000ng / mL (QC-H), 800ng / mL (QC-M), and 200ng / mL (QC-L) as quality control samples;

[0223] ④Use positive antibody and hook effect samples: dilute the positive antibody with NC to 2560ng / mL, 1280ng / mL, 640ng / mL, 320ng / mL, 160ng / mL, 80ng / mL, 40ng / mL, and 20ng / mL to prepare sensitivity samples of different concentrations. Dilute the positive antibody with NC samples to 10000ng / mL, 5000ng / mL, and 10ng / mL as hook effect samples.

[0224] 2) Methodological validation:

[0225] All samples are set up with duplicate wells. For QC-L, QC-M, QC-H, the OD value %CV of the duplicate wells of samples with SNR (Signal noise ratio) values ​​higher than the screening threshold must not exceed 20% (NC, screening threshold samples and samples with SNR (Signal noise ratio) values ​​lower than the screening threshold must not exceed 30%). If exceeded, the sample is considered invalid and should be eliminated in the result analysis.

[0226] In order to avoid the influence of external environmental factors on the measured values ​​of the analytical batch, this method uses SNR to replace all parameters of the OD value calculation method (NC samples are still calculated using OD values), and uses the median method to determine the OD value of each plate NC, SNR = Mean OD (verification sample or sample to be tested) / Median OD (NCs).

[0227] In the confirmation screening threshold and confirmation threshold tests, 30 animal samples were screened and confirmed in one analysis batch, and completed in 3 plates, with 15 individual samples and 3 NC, 3 QC-L, 3 QC-M, and 3 QC-H running on each plate, and the screening samples and confirmation samples were arranged alternately. Among the 15 individual samples, Biotin working solution was added as the undoped sample (i.e., the screening threshold sample), and the confirmation sample working solution was added as the doped sample (i.e., the confirmation threshold sample). At the same time, 1 set of NC, QC-L, QC-M, and QC-H were added with the confirmation sample working solution for confirmation analysis.

[0228] ① Screening threshold calculation:

[0229] After removing outliers, the (Log(SNR)) data were tested by SPSS software for SW test to determine normal distribution, and the screening threshold (SCP) and titer threshold (TCP) were calculated. The results are as follows: Fig.11 shown.

[0230] SW test p<0.05, skewness=0.487, its absolute value is less than 1, proving non-normality, but the data are not highly biased and have a long-tail distribution;

[0231] The SCP threshold is calculated as 1.055 by Log(SCP)=Median+1.645×(1.4826×MAD).

[0232] The TCP threshold is calculated as 1.125 by Log(TCP)=Median+3.09×(1.4826×MAD).

[0233] Note: Median is the median of the screening threshold sample Log(SNR); MAD is the absolute median difference of the screening threshold sample.

[0234] ② Calculation of confirmation threshold CCP:

[0235] Calculation of individual inhibition rate: inhibition rate % = 100% × (1-SNR value of drug-added sample value / SNR value of non-drug-added sample value);

[0236] The sample data was calculated using SPSS software to calculate CCP. The SW test was first performed. The results are as follows: Fig.12 shown.

[0237] SW test p>0.05, the absolute value of skewness is less than 1, proving normality. The confirmation threshold is calculated by the formula (CCP) = Mean + 2.33 × SD, and the confirmation threshold is 12.376%.

[0238] ③Sensitivity analysis:

[0239] Three batches of tests were performed using three NC, QC-L, QC-H samples and a sensitivity sample as one analysis batch. Linear fitting was performed using the two points above and below the screening threshold or titer threshold, and the concentration corresponding to the screening threshold or titer threshold was calculated using the interpolation method. The mean and SD of the six batches were calculated.

[0240] According to sensitivity = Mean + t0.05, df × SD, (t 0.05 : The constant corresponding to the 95% confidence interval in the t-test, df = n (灵敏度样本的总批数) -1. The sensitivity is reported as the concentration corresponding to the titer threshold. If no dilution produces a signal below the screening threshold (1.055) or the titer threshold (1.125), but reaches acceptable method sensitivity (≤100 ng / mL), then the sensitivity is reported as the lowest detection concentration, so the sensitivity of this experiment is 20 ng / ml. The statistical results of the three batches of experiments are shown in Table 6.

[0241] Table 6

[0242]

[0243] ④HOOK effect:

[0244] In the concentration range of 10-10000 ng / mL, as the concentration of anti-injectable recombinant asparaginase increased, the SNR value did not decrease, indicating that no Hook effect was found. The test results are shown in Table 7.

[0245] Table 7

[0246]

[0247]

[0248] 3. Sample testing:

[0249] The animal experimental samples after administration were taken for screening experimental analysis. The thresholds of SD rat serum at D-5 were all lower than the screening threshold, which was negative; at D14, samples 6 and 28 were higher than the screening threshold of 1.055 and were positive, and the rest were negative. Afterwards, the two samples were subjected to confirmation experiments, and their inhibition rates were all lower than the confirmation experiment threshold, and the results were negative. In summary, the prepared asparaginase has low immunogenicity. The specific data determined by the screening (D-5 results) are shown in Table 8.

[0250] Table 8

[0251] Sample ID SNR value Log(SNR) Sample ID SNR value Log(SNR) 1 0.98 -0.009 21 1.00 0.000 2 0.96 -0.018 22 0.97 -0.013 3 0.94 -0.027 23 0.95 -0.022 4 0.93 -0.032 24 0.97 -0.013 5 1.03 0.013 25 1.03 0.013 6 1.01 0.004 26 1.02 0.009 7 0.98 -0.009 27 0.97 -0.013 8 1.00 0.000 28 1.02 0.009 9 0.98 -0.009 29 1.00 0.000 10 0.97 -0.013 30 1.00 0.000 11 0.95 -0.022 31 1.00 0.000 12 0.94 -0.027 32 0.96 -0.018 13 1.13 0.053 33 1.06 0.025 14 1.05 0.021 34 1.08 0.033 15 1.01 0.004 35 1.02 0.009 16 0.94 -0.027 36 0.95 -0.022 17 0.96 -0.018 37 0.94 -0.027 18 0.92 -0.036 38 0.92 -0.036 19 0.92 -0.036 39 0.90 -0.046 20 1.04 0.017 40 1.00 0.000

[0252] The specific data of the screening determination (D14) is shown in Figure 9.

[0253] After screening and determination experiments, the sera of rats No. 6 and No. 28 were higher than the screening threshold of 1.055 and were positive, and the rest of the samples were negative.

[0254] Table 9

[0255]

[0256]

[0257] After further confirmation experiments, samples 6 and 28 were negative because their test results were less than the confirmation threshold of 12.376%.

[0258] The results of the confirmation experiment are shown in Table 10.

[0259] Sample ID SNR1 SNR2 Inhibition rate (%) 6 1.01 0.93 7.92 28 1.01 0.94 6.93

[0260] 4. Immunogenicity testing of other mutant proteins

[0261] According to the results of the immunogenicity test and validation experiment of Etb-1 preparation, animal experiments were conducted simultaneously with preparations Etb-2, Etb-3, Etb-4, Etb-5, Etb-6, Etb-7, Etb-8, and Etb-9 and wild-type asparaginase Etb-0. A solvent control group and a medium-dose group (6000 IU / kg) were set for each preparation sample. Animal immunogenicity analysis experiments were conducted using 6-9 week old SD rats (SPF grade, Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), male weight 262-290 g, female weight: 196-235 g. Five days before administration (D-5), the animals were randomly divided into 9 groups according to sex, with 5 males and 5 females in each group. The drug was administered once a day for 14 consecutive days, with a dosage volume of 10 ml / kg and a dosage rate of 2 ml / kg / min. The day of administration was defined as D1. Blood samples of the animals after administration were taken at D-14 and tested by ELISA. If the result was positive, a confirmation test was required. The results of D-14 are shown in Table 11.

[0262] Table 11

[0263] Negative number Positive number Positive rate (%) Vehicle control group 10 0 0 Etb-0 medium dose group 1 9 90 Etb-2 medium dose group 9 1 10 Etb-3 medium dose group 10 0 0 Etb-4 medium dose group 8 2 20 Etb-5 medium dose group 10 0 0 Etb-6 medium dose group 9 1 10 Etb-7 medium dose group 10 0 0 Etb-8 medium dose group 9 1 10 Etb-9 medium dose group 10 0 0

[0264] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A low immunogenic asparaginase mutant, characterized in that: The mutant is obtained by replacing T119 of wild-type asparaginase type II with C, K196 with A, Y218 with D, Y220 with A, and Y289 with R; the wild-type asparaginase type II is selected as the wild-type asparaginase of GenBank: WP_000394140.1, and its 326-position amino acid sequence is specifically: LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKP VVMVGAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKYGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY; the amino acid sequence of the asparaginase mutant is shown in SEQ ID No.

1.

2. A low immunogenic asparaginase mutant according to claim 1, characterized in that: The sequence encoding the amino acid is a polynucleotide sequence, and the polynucleotide sequence is shown in SEQ ID NO.

10.

3. A recombinant expression vector, characterized in that: Contains a polynucleotide sequence encoding the amino acid sequence of the low immunogenic asparaginase mutant according to claim 2.

4. A recombinant bacterium, characterized in that: The invention comprises the recombinant expression vector according to claim 3, wherein the recombinant bacteria is obtained by transforming the bacterial strain with the recombinant expression vector.

5. The recombinant bacterium according to claim 4, characterized in that The strain is Escherichia coli.

6. The recombinant bacterium according to claim 5, characterized in that The strain is the Escherichia coli BL21 (DE3) strain.

7. The method for preparing a low immunogenic asparaginase mutant according to claim 1 or 2, characterized in that: The following steps are involved: S1. Preparation of low immunogenic asparaginase mutant expression strain: The polynucleotide sequence SEQ ID NO.10 synthesized by the whole gene is used as a template, and primers are used for PCR amplification. The PCR product is purified, double-digested and inserted into a plasmid, and then transformed into Escherichia coli competent cells, positive clones are selected, and the plasmid is extracted and sequenced for verification. The plasmid is transformed into competent cells, and positive clones are selected to obtain an expression strain of a low-immunogenic asparaginase mutant; S2. Expression of low immunogenic asparaginase: The expression strain obtained in step S1 is fermented on LB medium and gradually expanded, and the bacterial cells are collected by centrifugation; S3. Purification of low immunogenic asparaginase: The sludge-like bacterial cells obtained in step S2 are disrupted with a lysis solution, and the supernatant is collected by centrifugation. The supernatant is eluted through a chromatography column, and the eluate is concentrated to obtain purified low-immunogenic asparaginase.

8. Use of the low immunogenic asparaginase mutant according to claim 1 or 2, the recombinant expression vector according to claim 3, the recombinant bacteria according to claims 4-6, and the low immunogenic asparaginase mutant prepared by the preparation method according to claim 7 in the preparation of a preparation for treating malignant tumors of the lymphatic system.

9. The use according to claim 8, characterized in that: The preparation is a lyophilized preparation with a low immunogenicity asparaginase mutant content of 20 g / L.

10. The use according to claim 9, characterized in that: The preparation also contains 0.38 g / L-1.02 g / L of anhydrous disodium hydrogen phosphate, 0.39 g / L-1.01 g / L of sodium dihydrogen phosphate monohydrate, 4.8 g / L of sodium chloride, 50 ppm of Tween 80 and 30 g / L-50 g / L of mannitol, and the pH value of the preparation is 6.5-7.

2.

11. Use of the low immunogenic asparaginase mutant according to claim 1 or 2, the recombinant expression vector according to claim 3, the recombinant bacteria according to claims 4-6, and the low immunogenic asparaginase mutant prepared by the preparation method according to claim 7 in the preparation of a preparation for treating acute lymphoblastic leukemia or lymphocytic lymphoma.

12. The use according to claim 11, characterized in that: The preparation is a lyophilized preparation with a low immunogenicity asparaginase mutant content of 20 g / L.

13. The use according to claim 12, characterized in that: The preparation also contains 0.38 g / L-1.02 g / L of anhydrous disodium hydrogen phosphate, 0.39 g / L-1.01 g / L of sodium dihydrogen phosphate monohydrate, 4.8 g / L of sodium chloride, 50 ppm of Tween 80 and 30 g / L-50 g / L of mannitol, and the pH value of the preparation is 6.5-7.2.

Images