Bst DNA polymerase mutant and use thereof

Abstract

Amino acid mutations at one or more different sites are carried out on the basis of a wild-type Bst-LF, and an obtained Bst DNA polymerase mutant has higher DNA polymerase activity, strand displacement activity, and / or thermal stability.

Description

Bst DNA polymerase mutants and their applications

[0001] Related applications

[0002] This application claims priority to Chinese patent applications filed on December 19, 2023, with application number CN202311743966.X entitled "Heat-resistant Bst DNA polymerase mutant and its application", and with application number CN202311743948.1 filed on December 19, 2023, entitled "Enzyme-enzyme-enhanced Bst DNA polymerase mutant and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of biotechnology and relates to a Bst DNA polymerase mutant and its application, and further relates to a Bst DNA polymerase mutant with high temperature resistance and / or enhanced enzyme activity and its application. Background Technology

[0004] Since Kornberg discovered and studied DNA polymerase I in detail from *Escherichia coli* in 1958, various DNA polymerases have been isolated and characterized from prokaryotes and eukaryotes, gaining widespread attention and application in the biomedical community. Large fragments of DNA polymerase derived from *Geobacillus stearothermophilus* (Bst) possess strong strand displacement activity and thermostability, exhibiting both 5'-3' DNA polymerase activity and lacking 5'-3' exonuclease activity. They are widely used in isothermal amplification experiments, such as multiple displacement amplification (MDA), rolling circle amplification (RCA), and loop-mediated isothermal amplification (LAMP). LAMP is an extremely powerful nucleic acid amplification method that allows for continuous amplification of target nucleic acids at a single temperature using 4-6 primers and a DNA polymerase with strand displacement activity. Similar to PCR, LAMP amplifies target nucleic acids exponentially. However, unlike PCR, LAMP does not require temperature heating for denaturation and cooling for annealing; primer binding and amplification occur at a single temperature, utilizing the strand displacement activity of DNA polymerase to achieve template denaturation. Therefore, LAMP can be performed without complex specialized laboratory equipment and is much faster, making it widely used in medical testing and the food industry. RT-LAMP is an extension of LAMP technology, directly using mRNA as a template for the LAMP reaction. It can use DNA polymerases with reverse transcription activity or multiple enzymes reacting together. Bst DNA polymerase, due to its good combination of DNA polymerase activity, strand displacement activity, and thermostability, has become the most mainstream enzyme in both LAMP and RT-LAMP technologies. In recent years, faster and more sensitive detection has become the mainstream trend, posing new challenges to the performance of Bst DNA polymerase.

[0005] Although LAMP and RT-LAMP technologies are widely used in many fields, some technical challenges remain. For example, the reaction temperature for LAMP and RT-LAMP is generally 60-65℃, which results in poor amplification of target nucleic acids with high GC content. A common strategy to address the difficulty of amplifying high GC content templates is to increase the reaction temperature, but wild-type Bst DNA polymerases with large fragments cannot withstand higher reaction temperatures. Furthermore, non-specific amplification in LAMP and RT-LAMP is also a technical challenge. Increasing the reaction temperature can reduce some non-specific amplification. Therefore, a heat-resistant Bst DNA polymerase is urgently needed to adapt to higher reaction temperatures and achieve better detection results.

[0006] In view of this, this application proposes a mutant Bst DNA polymerase with better thermostability and / or higher enzyme activity. Summary of the Invention

[0007] The purpose of this application is to provide a Bst DNA polymerase mutant with high thermal stability; and also to provide a Bst DNA polymerase mutant with higher enzyme activity than the truncated wild-type Bst DNA polymerase.

[0008] The technical solution adopted in this application is as follows:

[0009] A heat-resistant Bst DNA polymerase mutant, which is any one of the proteins described in a1-a4 below:

[0010] a1: A protein obtained by mutation based on the large fragment of wild-type Bst DNA polymerase with an amino acid sequence as shown in SEQ ID No. 1, wherein the mutation sites include any one or a combination of at least two of the following: F67, F81, A114, D119, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, S365, Y429, G430, R439, F445, E461, T487, T500, M502, N503, R553, and A565;

[0011] a2: A protein with essentially the same enzyme activity and thermal stability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence shown in a1.

[0012] a3: A protein that has more than 90% sequence homology with a1, and whose enzyme activity and thermal stability are basically equivalent to those of a1.

[0013] a4: A fusion protein obtained by linking a tag or restriction enzyme site to the N-terminus and / or C-terminus of any of the amino acid sequences described in a1-a3, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

[0014] In some embodiments, the mutation site includes any one or a combination of at least two of F67, A114, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, Y429, G430, R439, F445, E461, T487, M502, N503, R553, and A565.

[0015] In some implementations, the mutation is any one or a combination of at least two of the following: F67S, F81L, A114L, D119N, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, S365D or I, Y429W, G430E, R439C, F445L or I, E461K, T487N, T500A, M502V, N503S, R553H, and A565V. In some embodiments, the mutation is any one or a combination of at least two of the following: F67S, A114L, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, Y429W, G430E, R439C, F445L or I, E461K, T487N, M502V, N503S, R553H, and A565V. In some embodiments, the mutation may further be selected from any one or a combination of at least two of the following: F81L, D119N, S365D or I, and T500A.

[0016] In some embodiments, the protein is any one of the proteins described in b1-b3 below:

[0017] b1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1:

[0018] (1)V16R / I359L / E461K; (2)S65P / D428K; (3)R133Y / L320P; (4)A193M / N283L; (5)N204R / Q128G; (6)N283L; (7)A409P / N462K; (8)S65P / I359L; (9)L68K; (10)L68V; (11)A114L; (12)Q128G; (13)Q182E / L340R; (14)V2 81I; (15)V281L; (16)T321A / N503S; (17)S365D; (18)T487N; (19)F445L ;(20)F67S / F445L;(21)N204S / S365I;(22)I194V;(23)M33V / F81L / M50 2V; (24)M502V / R553H; (25)R339C; (26)D377G / R439C; (27)A565V; (28) N204K / T487N; (29) N204R / L320P / F445L; (30) Q128G / N204R / F445I; (31 )Q334C; (32) Q334H; (33) G430E; (34) D119N / R339H / I422T; (35) T500A;

[0019] b2: A protein with essentially the same enzyme activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence shown in b1.

[0020] b3: A fusion protein obtained by linking a tag or restriction enzyme site to the N-terminus and / or C-terminus of any of the amino acid sequences described in b1-b2, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

[0021] In some embodiments, the protein is any one of the following c1-c3:

[0022] c1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1:

[0023] (1)N204R / Q128G; (2)S65P / I359L; (3)F67S / F445L; (4)N204S / S365I; (5)R339C; (6 )D377G / R439C; (7)N204K / T487N; (8)N204R / L320P / F445L; (9)Q128G / N204R / F445I;

[0024] c2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than those mentioned above in the amino acid sequence shown in c1.

[0025] c3: A fusion protein obtained by linking a tag or restriction enzyme site to the N-terminus and / or C-terminus of any of the amino acid sequences described in c1-c2, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

[0026] In some embodiments, the tag is a His tag, and the enzyme cleavage site is a thrombin cleavage site.

[0027] A Bst DNA polymerase mutant with enhanced enzyme activity, comprising any one of the proteins described in d1-d4 below:

[0028] d1: A protein obtained by mutation based on the large fragment of wild-type Bst DNA polymerase with an amino acid sequence as shown in SEQ ID No. 1. The mutation sites include any one or a combination of at least two of the following: D119, Q128, R133, V281, L320, T321, T323, S328, R339, Q348, I359, Y429, R439, E461, K468, T487, N503, and E552.

[0029] d2: A protein with essentially the same enzyme activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence shown in d1.

[0030] d3: A protein that has more than 90% sequence homology with d1, and whose enzyme activity and thermostability are basically equivalent to those of d1.

[0031] d4: A fusion protein obtained by attaching a tag or restriction enzyme site to the N-terminus and / or C-terminus of any of the amino acid sequences described in d1-d3, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

[0032] In some embodiments, the mutation is any one or a combination of at least two of the following: D119N, Q128G, R133Y, V281I, L320P, T321A, T323A, S328V, R339C, Q348L, I359L, Y429W, R439C, E461K, K468E or N, T487N, N503S, E552G. In some embodiments, the mutation site includes any one or a combination of at least two of the following: Q128, R133, V281, L320, T321, T323, S328, R339, Q348, I359, Y429, R439, E461, K468, T487, N503, E552. In some implementations, the mutation is any one or a combination of at least two of the following: Q128G, R133Y, V281I, L320P, T321A, T323A, S328V, R339C, Q348L, I359L, Y429W, R439C, E461K, K468E, or N, T487N, N503S, E552G.

[0033] In some embodiments, the protein is any one of the following e1-e3:

[0034] e1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1:

[0035] (1)D119N / R339H / I422T; (2)S134P; (3)V16R / S328V; (4)A329D; (5)E96G / F171S / H278R / Q348L / E552G; (6 )L68K / K468E; (7) L68K / K468N; (8) L68K / I359L / D428K / Y429W; (9) I422T; (10) S134P / V143A; (11) T323A;

[0036] e2: A protein with essentially the same enzyme activity and thermal stability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence shown in e1.

[0037] e3: A fusion protein obtained by linking a tag or restriction enzyme site to the N-terminus and / or C-terminus of any of the amino acid sequences described in e1-e2, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

[0038] In some embodiments, the protein is any one of the following f1-f3:

[0039] f1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1:

[0040] (1)L68K / I359L / D428K / Y429W; (2)S134P / V143A; (3)S65P / I359L;

[0041] (4)A329D;

[0042] f2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than those mentioned above in the amino acid sequence shown in f1.

[0043] f3: A fusion protein obtained by linking a tag or restriction enzyme site to the N-terminus and / or C-terminus of any of the amino acid sequences described in f1-f2, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

[0044] A Bst DNA polymerase mutant is also provided, which is any one of the proteins described below g1-g4:

[0045] g1: A protein obtained by mutation based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1, wherein the mutation sites include any one or a combination of at least two of the following: F67, F81, A114, D119, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, S365, Y429, G430, R439, F445, E461, T487, T500, M502, N503, R553, A565, T323, S328, Q348, K468, and E552;

[0046] g2: A protein with essentially the same enzyme activity and thermal stability obtained by substituting, deleting, or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence shown in g1.

[0047] g3: A protein that shares over 90% sequence homology with g1, and whose enzyme activity and thermostability are essentially equivalent to those of g1; and,

[0048] g4: A fusion protein obtained by linking one or more tags or restriction sites to the N-terminus and C-terminus of any of the amino acid sequences described in g1-g3, wherein the tags or restriction sites do not affect the function of the Bst DNA polymerase mutant.

[0049] In some embodiments, the mutation sites include any one or a combination of at least two of the following: F67, A114, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, Y429, G430, R439, F445, E461, T487, M502, N503, R553, A565, T323, S328, Q348, K468, and E552. In some embodiments, the mutation sites include any one or a combination of at least two of the following: F67, A114, Q128, R133, I194, V281, L320, T321, Q334, R339, I359, Y429, G430, R439, F445, T487, N503, R553, A565, T323, S328, Q348, and K468.

[0050] In some embodiments, the mutation includes any one or a combination of at least two of the following: F67S, F81L, A114L, D119N, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, S365D or I, Y429W, G430E, R439C, F445L or I, E461K, T487N, T500A, M502V, N503S, R553H, A565V, T323A, S328V, Q348L, K468E or N, and E552G. In some embodiments, the mutation includes any one or a combination of at least two of the following: F67S, A114L, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, Y429W, G430E, R439C, F445L or I, E461K, T487N, M502V, N503S, R553H, A565V, T323A, S328V, Q348L, K468E or N, and E552G. In some embodiments, the mutation includes any one or a combination of at least two of the following: F67S, A114L, Q128G, R133Y, I194V, V281I or L, L320P, T321A, Q334C or H, R339C or H, I359L, Y429W, G430E, R439C, F445L or I, T487N, N503S, R553H, A565V, T323A, S328V, Q348L, K468E or N.

[0051] In some embodiments, the Bst DNA polymerase mutant is any one of the proteins described in h1-h3 below:

[0052] h1: A protein obtained by mutation of any combination of the following based on the large fragment of wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID No. 1:

[0053] (1)V16R / I359L / E461K; (2)S65P / D428K; (3)R133Y / L320P; (4)A193M / N283L; (5)N204R / Q128G; (6)N283L; (7)A409P / N462K; (8)S65P / I359L; (9)L68K; (10)L68V; (11)A114L; (12)Q128G; (13) Q182E / L340R; (14)V281I; (15)V281L; (16)T321A / N503S; (17)S365D; (18)T487N; (19)F445L; ( 20)F67S / F445L; (21)N204S / S365I; (22)I194V; (23)M33V / F81L / M502V; (24)M502V / R553H; (25) R339C; (26)D377G / R439C; (27)A565V; (28)N204K / T487N; (29)N204R / L320P / F445L; (30)Q128G / N204R / F445I; (31)Q334C; (32)Q334H; (33)G430E; (34)D119N / R339H / I422T; (35)T500A; (36)S 134P; (37)V16R / S328V; (38)A329D; (39)E96G / F171S / H278R / Q348L / E552G; (40)L68K / K468E; (41)L68K / K468N; (42)L68K / I359L / D428K / Y429W; (43)I422T; (44)S134P / V143A; and (45)T323A;

[0054] h2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations, based on the amino acid sequence shown in h1; and,

[0055] h3: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in h1-h2, wherein the tags or restriction enzyme sites do not affect the function of the Bst DNA polymerase mutant.

[0056] In some embodiments, the Bst DNA polymerase mutant is any one of the proteins described in i1-i3 below:

[0057] i1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1:

[0058] (1) N204R / Q128G; (2) S65P / I359L; (3) F67S / F445L; (4) N204S / S365I; (5) R339C; (6) D377G / R439C; (7) N204K / T487N; (8) N204R / L320P / F445L; (9) Q128G / N204R / F445I; (10) L68K / I359L / D428K / Y429W; (11) S134P / V143A; and (12) A329D;

[0059] i2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations, based on the amino acid sequence shown in i1; and,

[0060] i3: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in i1-i2, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

[0061] In some embodiments, the tag is a His tag, and the restriction enzyme site is a thrombin cleavage site. This application also discloses the encoding gene of the aforementioned Bst DNA polymerase mutant.

[0062] This application also discloses the expression vector of the aforementioned Bst DNA polymerase mutant.

[0063] This application also discloses the expression host bacteria of the aforementioned Bst DNA polymerase mutant.

[0064] This application also discloses the use of the above-mentioned Bst DNA polymerase mutant in MDA, RCA, LAMP or RT-LAMP.

[0065] This application also discloses a kit containing the above-mentioned Bst DNA polymerase mutant, which can be used for MDA, RCA, LAMP or RT-LAMP technologies.

[0066] This application involves amino acid mutations at one or more different sites on wild-type Bst-LF to obtain Bst DNA polymerase mutants with higher DNA polymerase activity and / or strand substitution activity. The DNA polymerase activity of the Bst DNA polymerase mutants can be increased by up to 216%, and the strand substitution activity by up to 255%. In the embodiments of this application, 65℃ RT-LAMP can be performed up to approximately 15 Ct values ​​earlier. The Bst DNA polymerase mutants with enhanced DNA polymerase activity and / or strand substitution activity of this application have superior application performance and can meet the needs for faster and more sensitive detection.

[0067] This application provides a Bst DNA polymerase mutant, derived from a truncated wild-type Bst DNA polymerase through amino acid mutations at one or more different sites. This mutant exhibits higher thermostability, with a Tm value significantly higher than the wild-type Bst DNA polymerase. Compared to the wild-type Bst DNA polymerase, the Bst DNA polymerase mutant of this application shows significantly increased residual activity after incubation at 65°C for 30 min, and can perform RT-LAMP reactions at a maximum temperature of 70°C. The improved thermostability of this Bst DNA polymerase mutant allows for a wider range of application temperatures, adapting to more complex application scenarios. The mutant of this application also includes improvements in one or at least two of the following properties: optimal reaction temperature, storage stability, transport stability, continuous synthesis capability, high GC template amplification capability, product specificity, and reaction sensitivity.

[0068] Compared with existing technologies, this application has the following advantages:

[0069] (1) This application can significantly improve the thermal stability of BstDNA polymerase by designing mutations at one or more different sites of amino acids. The protein melting temperature (Tm) is increased by more than 0.1℃, more than 0.2℃, more than 0.5℃, and more than 1℃ compared with the wild type; the increase can be more than 2℃, 3℃, and 4℃; and the increase can reach more than 5℃.

[0070] (2) The Bst DNA polymerase mutant of this application has improved thermal stability, and the residual activity can still reach more than 80% after incubation at 65°C for 30 min.

[0071] (3) The Bst DNA polymerase mutant of this application can work in high-temperature RT-LAMP and can complete the RT-LAMP reaction at 70°C.

[0072] (4) This application obtains Bst DNA polymerase mutants by designing mutations at one or more different sites of amino acids. The DNA polymerase activity and / or strand substitution activity are improved compared with wild type. The DNA polymerase activity can be increased by up to 216%, and the strand substitution activity can be increased by up to 120% to 255%.

[0073] (5) The Bst DNA polymerase mutant of this application has improved performance in LAMP and / or RT-LAMP, and can significantly shorten the elution time in RT-LAMP at 65℃, with a smaller Ct value, up to 15 Ct values ​​earlier.

[0074] (6) The Bst DNA polymerase mutant of this application has improved reaction sensitivity, thermal stability and other properties. Attached Figure Description

[0075] Figure 1 shows a comparison of ΔTm between Bst-LF and Bst DNA polymerase mutants in one embodiment of this application.

[0076] Figure 2 shows a comparison of the residual activity of Bst-LF and Bst DNA polymerase mutants after incubation at 65°C for 30 min in one embodiment of this application.

[0077] Figure 3 shows the RT-LAMP curve of Bst-LF at 68°C in one embodiment of this application.

[0078] Figure 4 shows the RT-LAMP curve of ME5 at 68°C in one embodiment of this application.

[0079] Figure 5 shows the RT-LAMP curve of ME8 at 68°C in one embodiment of this application.

[0080] Figure 6 shows the RT-LAMP curve of ME20 at 68°C in one embodiment of this application.

[0081] Figure 7 shows the RT-LAMP curve of ME21 at 68°C in one embodiment of this application.

[0082] Figure 8 shows the RT-LAMP curve of ME25 at 68°C in one embodiment of this application.

[0083] Figure 9 shows the RT-LAMP curve of ME26 at 68°C in one embodiment of this application.

[0084] Figure 10 shows the RT-LAMP curve of ME28 at 68°C in one embodiment of this application.

[0085] Figure 11 shows the RT-LAMP curve of ME29 at 68°C in one embodiment of this application.

[0086] Figure 12 shows the RT-LAMP curve of ME30 at 68°C in one embodiment of this application.

[0087] Figure 13 shows the RT-LAMP curve of ME28 at 70°C in one embodiment of this application.

[0088] Figure 14 shows the RT-LAMP curve of ME29 at 70°C in one embodiment of this application.

[0089] Figure 15 shows the RT-LAMP curve of ME30 at 70°C in one embodiment of this application.

[0090] Figure 16 shows a comparison of DNA polymerase activities between Bst-LF and Bst DNA polymerase mutant in one embodiment of this application.

[0091] Figure 17 shows a comparison of strand displacement activities between Bst-LF and Bst DNA polymerase mutant in one embodiment of this application.

[0092] Figure 18 shows a comparison of Bst-LF, ME9, and ME51 at 65°C RT-LAMP in one embodiment of this application.

[0093] Figure 19 shows a comparison of Bst-LF with ME12, ME16, and ME54 at 65°C in one embodiment of this application.

[0094] Figure 20 shows a comparison of Bst-LF and ME26 at 65°C RT-LAMP in one embodiment of this application. Detailed Implementation

[0095] The specific embodiments of this application will be further described below with reference to the accompanying drawings, but the description of the embodiments does not limit the scope of protection of this application in any way.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to limit this application.

[0097] The description of mutations in this application is a description of mutations recognized by those skilled in the art. For example, F67S, a mutation at one of the sites, refers to the mutation of phenylalanine (F) at position 67 of the amino acid sequence shown in SEQ ID NO:1 to serine (S), that is, phenylalanine (F) at position 67 is replaced by serine (S).

[0098] The "homology" (sequence identity percentage) of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment, and, where necessary, the introduction of vacancies to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the sequence identity percentage (%) of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence by the total number of amino acid residues (or bases) in the candidate or reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. Publicly available tools can be used, for example, such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Molecular Biology 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), and ClustalW2 (available on the website of the European Bioinformatics Institute). Those skilled in the art can use the default parameters provided by the tools or can appropriately customize the parameters as needed for the comparison, for example by selecting a suitable algorithm.

[0099] As used in this application, the term "amino acid" refers to an organic compound that includes amino (-NH2) and carboxyl (-COOH) functional groups, as well as the side chain characteristic of each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, summarized below.

[0100] In this application, unless otherwise stated, "nucleic acid" has the well-known meaning in the art and is also called "polynucleotide," which is a molecule formed by multiple nucleotide monomers.

[0101] Unless otherwise specified, the term "vector" in this application refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element. Vectors can be, for example, plasmids, granules, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), RNA vectors, or linear or circular DNA or RNA molecules, which may include chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecules. This term includes vectors that serve as self-replicating nucleic acid structures as well as vectors incorporated into the host cell genome. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked.

[0102] Unless otherwise specified, the term "expression host bacteria" in this application may be used interchangeably with "host cell".

[0103] Host cells can include bacterial, fungal, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; Bacillus family members such as *Bacillus subtilis*; *Streptococcus pneumoniae*; and *Haemophilus influenzae*. Suitable fungi include *Saccharomyces cerevisiae* and *Pichia pastoris*. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0104] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.

[0105] Example 1

[0106] This embodiment provides a thermostable Bst DNA polymerase mutant, and its vector construction, expression, and purification method.

[0107] The wild-type Bst DNA polymerase was obtained by truncating DNA polymerase I from Geobacillus stearothermophilus and is named Bst-LF in this application. The amino acid sequence of Bst-LF is shown in SEQ ID NO.1, and the nucleic acid sequence encoding Bst-LF is shown in SEQ ID NO.2.

[0108] The thermostable Bst DNA polymerase mutant provided in this embodiment comprises replacing, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.1, forming a mutant protein with similar function to SEQ ID NO.1. The mutation sites of the Bst DNA polymerase mutant are detailed in Table 1.

[0109] Table 1 Bst DNA polymerase mutants

[0110] In this embodiment, the nucleic acid sequences encoding Bst-LF and the Bst DNA polymerase mutants in Table 1 can be obtained using point mutation PCR or gene synthesis. If necessary, the nucleic acid sequences can be optimized using E. coli codons. The obtained nucleic acid sequences correctly encoding Bst DNA polymerase are ligated into the vector pET28a via double digestion with Nde I and Xho I or homologous recombination. The N-terminus of Bst-LF and its mutants is linked to a His tag via a thrombin cleavage site. The vector carrying the Bst DNA polymerase encoding sequence is then transformed into the expression host *E. coli* Rosetta.

[0111] Engineered bacteria containing the Bst DNA polymerase mutant encoding a nucleic acid sequence were cultured at 37°C until OD200. 600 When the pH is 0.7–0.8, 0.5 mM IPTG is added for induction, and then the culture is continued at 16°C for 16–20 hours. The fermented E. coli cells are collected by centrifugation, then the cells are resuspended and sonicated, purified by nickel column chromatography, and finally concentrated and replaced with stock solution to obtain purified Bst-LF and Bst DNA polymerase mutant proteins.

[0112] The thermostability-enhanced Bst DNA polymerase mutant described in this application is not limited to the above-mentioned vector, host, culture, and purification conditions. Any method that can obtain Bst DNA polymerase mutant proteins with normal structure and function is also applicable to this application.

[0113] Example 2

[0114] The DNA polymerase activity of the Bst-LF and Bst DNA polymerase mutants obtained in Example 1 was tested.

[0115] Experimental Principle: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 1 were diluted to a certain concentration using stock solutions for DNA polymerase activity testing. In this example, the template and primers used to evaluate the DNA polymerase activity of the Bst DNA polymerase mutant were derived from the same single-stranded deoxynucleotide sequence, which is named Pol-PT in this application. The sequence of this single strand is: 5'-ACAACCATTTATGTAGCATTTATGAAATTTTTAAATCAATTTACTATTGGCTACTGCATACGCGAAAGCGTATGCAGTAGCC-3' (Seq ID No. 3). After annealing, the Pol-PT single strand forms a monocyclic substrate, which serves as both the template and primer for the DNA polymerization reaction. In the reaction system, Bst DNA polymerase extends the monocyclic nucleic acid substrate into a double-stranded structure at 37°C for 5 min. The double-strand yield was then measured using dsDNA nucleic acid dye to evaluate the Bst DNA polymerase activity. DNA polymerase activity of Bst DNA polymerase was compared with wild-type Bst-LF as a control.

[0116] The detailed reaction procedure and system are as follows: First, prepare the primer annealing system according to Table 2, incubate the prepared primer annealing system in a 95℃ metal bath for 2 minutes, and then let it stand on ice for 5 minutes. Then, prepare the polymerization reaction system according to Table 3, and incubate the prepared polymerization reaction system in a 37℃ metal bath for 5 minutes. After the reaction, prepare the dsDNA nucleic acid dye (Yisheng product Cat#12641) and reaction solution according to the instructions using a completely black 96-well microplate. After incubating at room temperature for 5 minutes, measure the fluorescence value under the conditions of Ex / Em = 480nm / 520nm.

[0117] Table 2 Primer annealing system

[0118] Table 3 Polymerization Reaction System

[0119] The DNA polymerase activity of the Bst DNA polymerase mutant was compared with that of wild-type Bst-LF. The results are shown in Table 4, indicating that the obtained Bst DNA polymerase has basic polymerization activity.

[0120] Table 4 DNA polymerase activities of Bst-LF and Bst DNA polymerase mutants

[0121] Example 3

[0122] The strand substitution activity of the Bst-LF and Bst DNA polymerase mutants obtained in Example 1 was tested.

[0123] Experimental Principle: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 1 were diluted to a certain concentration using stock solutions and then subjected to strand displacement activity testing. In this example, the template and primers used to evaluate the strand displacement activity of the Bst DNA polymerase mutant were derived from the same single-stranded deoxynucleotide sequence. The 5' end of this single strand was modified with the fluorescent group FAM, and this single strand was named Strand F, with the nucleic acid sequence: 5'-ACAACCATTTATGTAGCATTTATGAAATTTTTAAATCAATTTACTATTGGCTACTGCATACGCGAAAGCGTATGCAGTAGCC-3' (SEQ ID NO. 3). A complementary primer for the 5' end of Strand F was also designed, named Strand Q. The 3' end of Strand Q was modified with the quencher group BHQ, and the sequence of Strand Q was: 5'-TAAAAATTTCATAAATGCTACATAAATGGTTGT-3' (SEQ ID NO. 4). Following the annealing reaction, Strand F forms a single-stranded circular substrate, while Strand Q binds complementary to the 5' end of Strand F. The quenching group carried by Strand Q quenches the fluorescent group carried by Strand F. In the reaction system, Bst DNA polymerase briefly extends the single-stranded nucleic acid substrate Strand F at 37°C for 5 min. Then, the strand displacement activity of Bst DNA polymerase displaces Strand Q from Strand F, releasing it into the solution, allowing the fluorescent group carried by Strand F to emit light. The fluorescence signal is then collected to characterize the strength of the strand displacement activity. The strand displacement activity of Bst DNA polymerase is compared with wild-type Bst-LF as a control.

[0124] The detailed reaction procedure and system are as follows: First, prepare the primer annealing system according to Table 5, incubate the prepared primer annealing system in a 95℃ metal bath for 2 minutes, and then let it stand on ice for 5 minutes. Then, prepare the chain displacement reaction system according to Table 6, and incubate the prepared chain displacement reaction system in a 37℃ metal bath for 5 minutes. After the reaction is complete, take 20 μL of the reaction solution into a completely black 96-well microplate, add 80 μL of DEPC H2O, vortex to mix, and then measure the fluorescence value under the conditions of Ex / Em = 485nm / 535nm.

[0125] Table 5 Primer annealing system

[0126] Table 6 Chain displacement reaction system

[0127] The strand substitution activity of the Bst DNA polymerase mutant was compared with that of wild-type Bst-LF. The results are shown in Table 7, indicating that the obtained Bst DNA polymerase mutant has basic strand substitution activity.

[0128] Table 7 Strand displacement activities of Bst-LF and Bst DNA polymerase mutants

[0129] Example 4

[0130] The melting temperature (Tm) of the Bst-LF and Bst DNA polymerase mutants obtained in Example 1 was determined.

[0131] In this embodiment, the thermal stability of Bst DNA polymerase is reflected by measuring the melting temperature (Tm) of the Bst DNA polymerase protein.

[0132] The detailed principle is as follows: when a protein peptide chain folds in water, its hydrophilic groups wrap around the hydrophobic groups. Under external pressure (high temperature), the protein denatures and unfolds, gradually exposing the hydrophobic groups. At this time, the hydrophobic fluorescent dye in the system will bind to the hydrophobic groups of the protein and emit fluorescence. The Tm of different proteins can be calculated by the change of fluorescence curve, thereby enabling the comparison of the thermal stability of mutants.

[0133] The detailed method is as follows: The Bst DNA polymerase protein purified in Example 1 is diluted to an appropriate concentration. The hydrophobic fluorescent dye is mixed with the diluted protein solution. In a real-time quantitative PCR instrument, the temperature is increased to 35°C at a constant rate of 1.6°C / s and held for 20s. Then, the temperature is gradually increased to 80°C and held for 1s. During the temperature increase, the fluorescence signal is collected every 0.5s. Tm is calculated based on the collected signal analysis.

[0134] The Bst DNA polymerase Tm assay described in this application is not limited to the above method; other methods applicable to protein Tm assay are also applicable to this application.

[0135] The Bst DNA polymerase mutant with improved thermal stability obtained in this application exhibits a significantly increased melting temperature (Tm) compared to the wild-type Bst-LF (Figure 1). Specifically, the increase is ≥0.1℃, ≥0.2℃, ≥0.5℃, and ≥1.0℃ compared to the wild type; more significant increases are seen at ≥2.0℃, ≥3.0℃, and ≥4.0℃; with the largest increase exceeding 5.0℃. To a certain extent, melting temperature (Tm) reflects the thermal stability of Bst DNA polymerase; a higher melting temperature (Tm) indicates better thermal stability. This demonstrates that the Bst DNA polymerase mutant obtained in this application possesses better thermal stability than Bst-LF.

[0136] Example 5

[0137] The residual activity of the Bst-LF and Bst DNA polymerase mutants obtained in Example 1 was detected after treatment at 65°C for 30 minutes.

[0138] Detailed Method: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 1 were diluted to a certain concentration with stock solution and incubated at 65°C for 30 min before DNA polymerase activity testing. A control was prepared by directly testing the DNA polymerase activity of the same Bst DNA polymerase without 30 min of 65°C heat incubation. The ratio of DNA polymerase activity after heat incubation to that before heat incubation is the percentage of residual activity of the Bst DNA polymerase. In this example, the method for detecting the DNA polymerase activity of the Bst DNA polymerase mutant before and after heat incubation is consistent with that in Example 2. The results show that the residual activity of the Bst DNA polymerase mutant obtained in this application is significantly improved compared with the wild type after incubation at 65°C for 30 min (Figure 2). In detail, the residual activity of mutation numbers ME1~ME8, ME16, ME18~ME31, and ME34 is significantly improved, with ME5, ME8, ME28, ME29, and ME30 having residual activities of over 50%, and the highest residual activities of ME8 and ME29 reaching over 80%.

[0139] Example 6: RT-LAMP reaction at 68℃ using the mutant obtained in Example 1.

[0140] The effectiveness of the Bst DNA polymerase mutant obtained in Example 1 in the RT-LAMP reaction at 68°C was tested.

[0141] In this embodiment, the RT-LAMP testing method uses Yisheng's "RT-LAMP Dye Assay Kit (UDG plus)" (Cat#13762) and "RT-LAMP COVID-19 Primer Master Mix (N)" (Cat#13966). For detailed operating steps, please refer to the official website instructions.

[0142] Combining Examples 2 and 3, mutants with excellent thermal stability were selected for 68℃ RT-LAMP reaction, with each mutant undergoing at least 8 replicates. The test results of 68℃ RT-LAMP are shown in Figure 3-12.

[0143] Results: As shown in Figure 3-12, Bst-LF cannot perform the RT-LAMP reaction at 68℃. However, the Bst DNA polymerase mutants ME5, ME8, ME20, ME21, ME25, ME26, ME28, ME29 and ME30 with improved thermal stability described in this application have improved thermal stability than Bst-LF. Except for ME26, which is not very suitable for 68℃ RT-LAMP, the others can complete 68℃ RT-LAMP very well.

[0144] Example 7: RT-LAMP reaction at 70°C using the mutant obtained in Example 1.

[0145] The effectiveness of the Bst DNA polymerase mutant obtained in Example 1 in the RT-LAMP reaction at 70°C was tested.

[0146] In this embodiment, the RT-LAMP testing method uses Yisheng's "RT-LAMP Dye Assay Kit (UDG plus)" (Cat#13762) and "RT-LAMP COVID-19 Primer Master Mix (N)" (Cat#13966). For detailed operating steps, please refer to the official website instructions.

[0147] Based on Examples 2, 3, and 4, mutants with excellent thermal stability were selected for 70℃ RT-LAMP reactions, with each mutant undergoing at least 8 replicates. The test results of 70℃ RT-LAMP are shown in Figures 13-15.

[0148] Results: The thermostability-enhanced Bst DNA polymerase mutants ME5, ME8, ME20, ME21, ME25, ME26, ME28, ME29, and ME30 described in this application exhibited significant fluorescence enhancement peaks in the 70℃ RT-LAMP test. ME29 and ME30 showed the best performance, indicating that ME29 and ME30 possess significantly improved thermostability and can successfully complete the RT-LAMP reaction at 70℃.

[0149] In summary, amino acid mutations at one or more different sites can significantly improve the thermostability of Bst DNA polymerase and substantially increase the Tm of the Bst DNA polymerase mutant. Compared to wild-type Bst DNA polymerase, the residual activity after incubation at 65°C for 30 min is significantly improved, and it can perform RT-LAMP reactions at up to 70°C. The thermostability-enhanced Bst DNA polymerase of this application has a wider range of application temperatures and can adapt to more complex application scenarios.

[0150] Example 8

[0151] This embodiment provides a Bst DNA polymerase mutant with enhanced DNA polymerase activity, and a method for constructing, expressing, and purifying its vector.

[0152] The wild-type Bst DNA polymerase was obtained by truncating DNA polymerase I from Geobacillus stearothermophilus and is named Bst-LF in this application. The amino acid sequence of Bst-LF is SEQ ID NO.1, and the nucleic acid sequence encoding Bst-LF is SEQ ID NO.2.

[0153] The Bst DNA polymerase mutant with enhanced activity provided in this embodiment is formed by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.1, resulting in a mutant protein with similar function to SEQ ID NO.1. The mutation sites of the Bst DNA polymerase mutant are detailed in Table 8.

[0154] Table 8 Bst DNA polymerase mutants

[0155] In this embodiment, the nucleic acid sequences encoding Bst-LF and the Bst DNA polymerase mutant in Table 8 can be obtained by point mutation PCR or gene synthesis. If necessary, the nucleic acid sequences can be optimized using *E. coli* codons. The obtained nucleic acid sequence encoding the correct Bst DNA polymerase is ligated into the vector pET28a via double digestion with Nde I and Xho I or homologous recombination. The N-terminus of Bst-LF and its mutant is linked to a His tag via a thrombin cleavage site. The vector containing the correct Bst DNA polymerase encoding sequence is then transformed into the expression host *E. coli* Rosetta. The engineered bacteria containing the Bst DNA polymerase mutant encoding nucleic acid sequence are cultured at 37°C until OD200. 600 When the pH is 0.7–0.8, 0.5 mM IPTG is added for induction, and then the culture is continued at 16°C for 16–20 hours. The fermented E. coli cells are collected by centrifugation, then the cells are resuspended and sonicated, purified by nickel column chromatography, and finally concentrated and replaced with stock solution to obtain purified Bst-LF and Bst DNA polymerase mutant proteins.

[0156] The Bst DNA polymerase mutant with enhanced DNA polymerase activity described in this application is not limited to the above-mentioned vector, host, culture and purification conditions. Any method that can obtain Bst DNA polymerase mutant protein with normal structure and function is also applicable to this application.

[0157] Example 9

[0158] The DNA polymerase activity of the Bst-LF and Bst DNA polymerase mutants obtained in Example 8 was detected.

[0159] Experimental Principle: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 8 were diluted to a certain concentration using stock solution for DNA polymerase activity testing. In this example, the template and primers used to evaluate the DNA polymerase activity of the Bst DNA polymerase mutant were derived from the same single-stranded deoxynucleotide sequence, which is named Pol-PT in this application. The sequence of this single strand is: 5'-ACAACCATTTATGTAGCATTTATGAAATTTTTAAATCAATTTACTATTGGCTACTGCATACGCGAAAGCGTATGCAGTAGCC-3' (SEQ ID NO.3). After annealing, the Pol-PT single strand forms a monocyclic substrate, which serves as both the template and primer for the DNA polymerization reaction. In the reaction system, Bst DNA polymerase extends the monocyclic nucleic acid substrate into a double-stranded structure at 37°C for 5 min. The double-strand yield was then measured using dsDNA nucleic acid dye to evaluate the Bst DNA polymerase activity. DNA polymerase activity of Bst DNA polymerase was compared with wild-type Bst-LF as a control.

[0160] The detailed reaction procedure and system are as follows: First, prepare the primer annealing system according to Table 9, incubate the prepared primer annealing system in a 95℃ metal bath for 2 minutes, and then let it stand on ice for 5 minutes. Then, prepare the polymerization reaction system according to Table 3, and incubate the prepared polymerization reaction system in a 37℃ metal bath for 5 minutes. After the reaction, prepare the dsDNA nucleic acid dye (Yisheng product Cat#12641) and reaction solution according to the instructions using a completely black 96-well microplate. After incubating at room temperature for 5 minutes, measure the fluorescence value under the conditions of Ex / Em = 480nm / 520nm.

[0161] Table 9 Primer Annealing System

[0162] Table 10 Polymerization Reaction System

[0163] The DNA polymerase activities of Bst DNA polymerase mutants were compared with wild-type Bst-LF as a control. The results are shown in Table 11 and Figure 16. The DNA polymerase activities of mutants ME3 and ME50 were increased by more than 20%, those of mutants ME26 and ME44 were increased by more than 40%, those of mutants ME8, ME9, ME12 and ME16 were increased by more than 60%, those of mutant ME51 were increased by more than 80%, and those of mutant ME54 were increased by 115%.

[0164] Table 11 DNA polymerase activities of Bst-LF and Bst DNA polymerase mutants

[0165] Example 10

[0166] The strand displacement activity of the Bst-LF and Bst DNA polymerase mutants obtained in Example 8 was tested.

[0167] Experimental Principle: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 8 were diluted to a certain concentration using stock solution and then subjected to strand substitution activity testing. In this example, the template and primers used to evaluate the strand substitution activity of the Bst DNA polymerase mutant were derived from the same single-stranded deoxynucleotide sequence. The 5' end of this single strand was modified with the fluorescent group FAM, and this single strand was named Strand F, with the sequence: 5'-ACAACCATTTATGTAGCATTTATGAAATTTTTAAATCAATTTACTATTGGCTACTGCATACGCGAAAGCGTATGCAGTAGCC-3' (SEQ ID NO.3). A complementary primer for the 5' end of Strand F was also designed, named Strand Q. The 3' end of Strand Q was modified with the quencher group BHQ, and the sequence of Strand Q was: 5'-TAAAAATTTCATAAATGCTACATAAATGGTTGT-3' (SEQ ID NO.4). Following the annealing reaction, Strand F forms a single-stranded circular substrate, while Strand Q binds complementary to the 5' end of Strand F. The quenching group carried by Strand Q quenches the fluorescent group carried by Strand F. In the reaction system, Bst DNA polymerase briefly extends the single-stranded nucleic acid substrate Strand F at 37°C for 5 min. Then, the strand displacement activity of Bst DNA polymerase displaces Strand Q from Strand F, releasing it into the solution, allowing the fluorescent group carried by Strand F to emit light. The fluorescence signal is then collected to characterize the strength of the strand displacement activity. The strand displacement activity of Bst DNA polymerase is compared with wild-type Bst-LF as a control.

[0168] The detailed reaction procedure and system are as follows: First, prepare the primer annealing system according to Table 12, incubate the prepared primer annealing system in a 95℃ metal bath for 2 minutes, and then let it stand on ice for 5 minutes. Then, prepare the chain displacement reaction system according to Table 13, and incubate the prepared chain displacement reaction system in a 37℃ metal bath for 5 minutes. After the reaction is complete, take 20 μL of the reaction solution into a completely black 96-well microplate, add 80 μL of DEPC H2O, vortex to mix, and then measure the fluorescence value under the conditions of Ex / Em = 485nm / 535nm.

[0169] Table 12 Primer Annealing System

[0170] Table 13 Chain displacement reaction system

[0171] The strand substitution activities of Bst DNA polymerase mutants were compared with wild-type Bst-LF as a control. The results are shown in Table 14 and Figure 17. The strand substitution activities of mutants ME1, ME2, ME3, ME7, ME14, ME34, ME50, ME54, and ME55 were increased by more than 20%, those of mutants ME9, ME40, ME43, and ME46 were increased by more than 40%, those of mutant ME16 were increased by more than 60%, those of mutant ME26 were increased by more than 80%, those of mutant ME12 were increased by 121%, and those of mutant ME51 were increased by 155%.

[0172] Table 14 Strand displacement activities of Bst-LF and Bst DNA polymerase mutants

[0173] Example 11

[0174] The mutant obtained in Example 8 was subjected to a 65°C RT-LAMP reaction.

[0175] The effect of the Bst DNA polymerase mutant obtained in Example 8 on the RT-LAMP reaction at 65°C was tested.

[0176] In this embodiment, the RT-LAMP testing method uses Yisheng's "RT-LAMP Dye Assay Kit (UDG plus)" (Cat#13762) and "RT-LAMP COVID-19 Primer Master Mix (N)" (Cat#13966). Detailed operating procedures can be found in the official website's instruction manual. To better compare the differences between Bst DNA polymerase mutants, the amount of template input can be appropriately reduced.

[0177] In conjunction with Examples 9 and 10, mutants exhibiting superior DNA polymerase activity and / or strand displacement activity were selected for 65°C RT-LAMP reactions, with each mutant undergoing at least 4 or 8 replicates. The results of the 65°C RT-LAMP tests are shown in Figures 18-20.

[0178] Results: As shown in Figures 18-20, ME9, ME12, ME16, ME26, ME51, and ME54 exhibited better performance than Bst-LF in RT-LAMP at 65℃, with earlier elution times and similar fluorescence peaks. Among them, ME12, ME51, and ME54 showed relatively similar performance, exhibiting the best performance among all mutants, advancing the peak time by up to approximately 15 Ct values. ME9, ME16, and ME26 were second best, but still performed better than Bst-LF, advancing the peak time by approximately 8 Ct values. This indicates that the increased DNA polymerase activity and / or strand displacement activity of Bst DNA polymerase mutants can improve the performance of LAMP or RT-LAMP, resulting in earlier elution times. It is reasonable to speculate that these Bst DNA polymerase mutants with enhanced activity may have higher sensitivity, enabling the detection of even minute amounts of template.

[0179] In summary, this application involves amino acid mutations at one or more different sites on wild-type Bst-LF to obtain Bst DNA polymerase mutants with higher DNA polymerase activity and / or strand substitution activity. The DNA polymerase activity of the Bst DNA polymerase mutants can be increased by up to 216%, and the strand substitution activity by up to 255%. In the embodiments of this application, 65℃ RT-LAMP can be performed up to approximately 15 Ct values ​​earlier. The Bst DNA polymerase mutants with enhanced DNA polymerase activity and / or strand substitution activity of this application exhibit superior application performance and can meet the needs for faster and more sensitive detection.

[0180] The applicant declares that this application illustrates the detailed method of this application through the above embodiments, but this application is not limited to the above detailed method, that is, it does not mean that this application must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of the raw materials of this application's product, addition of auxiliary components, selection of detailed methods, etc., all fall within the protection scope and disclosure scope of this application.

Claims

1. A thermostable Bst DNA polymerase mutant, comprising any one of the proteins described in a1-a4 below: a1: A protein obtained by mutation based on the large fragment of wild-type Bst DNA polymerase with an amino acid sequence as shown in SEQ ID No. 1, wherein the mutation sites include any one or a combination of at least two of the following: F67, F81, A114, D119, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, S365, Y429, G430, R439, F445, E461, T487, T500, M502, N503, R553, and A565; a2: A protein with essentially the same enzyme activity and thermal stability obtained by substituting, deleting, or adding one or more amino acid residues other than the aforementioned mutations into the amino acid sequence shown in a1. a3: A protein that shares over 90% sequence homology with a1, and whose enzyme activity and thermostability are essentially equivalent to those of a1; and, a4: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in a1-a3, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

2. The Bst DNA polymerase mutant according to claim 1, wherein, The mutation is any one or a combination of at least two of the following: F67S, F81L, A114L, D119N, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, S365D or I, Y429W, G430E, R439C, F445L or I, E461K, T487N, T500A, M502V, N503S, R553H, and A565V.

3. The Bst DNA polymerase mutant according to claim 1, wherein it is any one of the proteins described in b1-b3 below: b1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1: (1)V16R / I359L / E461K; (2)S65P / D428K; (3)R133Y / L320P; (4)A193M / N283L; (5) N204R / Q128G; (6) N283L; (7)A409P / N462K; (8)S65P / I359L; (9)L68K; (10)L68V; (11)A114L; (12)Q128G; (13)Q182E / L340R; (14)V281I; (15)V281L; (16)T321A / N503S; (17)S365D; (18)T487N; (19)F445L; (20)F67S / F445L; (21) N204S / S365I; (22)I194V; (23)M33V / F81L / M502V; (24)M502V / R553H; (25)R339C; (26)D377G / R439C; (27)A565V; (28)N204K / T487N; (29)N204R / L320P / F445L; (30)Q128G / N204R / F445I; (31)Q334C; (32)Q334H; (33)G430E; (34)D119N / R339H / I422T; and, (35)T500A; b2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations, based on the amino acid sequence shown in b1; and, b3: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in b1-b2, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

4. The Bst DNA polymerase mutant according to claim 1, wherein it is any one of the proteins described in c1-c3 below: c1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1: (1) N204R / Q128G; (2)S65P / I359L; (3)F67S / F445L; (4) N204S / S365I; (5)R339C; (6)D377G / R439C; (7) N204K / T487N; (8) N204R / L320P / F445L; and, (9)Q128G / N204R / F445I; c2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than those described above in the amino acid sequence shown in c1; and, c3: A fusion protein obtained by linking one or more tags or restriction sites to the N-terminus and C-terminus of any of the amino acid sequences described in c1-c2, wherein the tags or restriction sites do not affect the function of the Bst DNA polymerase mutant.

5. A Bst DNA polymerase mutant with enhanced enzyme activity, comprising any one of the proteins described in d1-d4 below: d1: A protein obtained by mutation based on the large fragment of wild-type Bst DNA polymerase with an amino acid sequence as shown in SEQ ID No.

1. The mutation sites include any one or a combination of at least two of the following: D119, Q128, R133, V281, L320, T321, T323, S328, R339, Q348, I359, Y429, R439, E461, K468, T487, N503, and E552. d2: A protein with essentially the same enzyme activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence shown in d1. d3: A protein that has more than 90% sequence homology with d1, and whose enzyme activity and thermostability are basically equivalent to those of d1. as well as, d4: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in d1-d3, wherein the tags or restriction enzyme sites do not affect the function of the Bst DNA polymerase mutant.

6. The Bst DNA polymerase mutant of claim 5, wherein, The mutation is any one or a combination of at least two of the following: D119N, Q128G, R133Y, V281I, L320P, T321A, T323A, S328V, R339C, Q348L, I359L, Y429W, R439C, E461K, K468E or N, T487N, N503S, E552G.

7. The Bst DNA polymerase mutant according to claim 5, wherein it is any one of the proteins described in e1-e3 below: e1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1: (1)D119N / R339H / I422T; (2)S134P; (3) V16R / S328V; (4)A329D; (5)E96G / F171S / H278R / Q348L / E552G; (6)L68K / K468E; (7)L68K / K468N; (8)L68K / I359L / D428K / Y429W; (9)I422T; (10)S134P / V143A; and, (11)T323A; e2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations, based on the amino acid sequence shown in e1; and, e3: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in e1-e2, wherein the tags or restriction enzyme sites do not affect the function of the Bst DNA polymerase mutant.

8. The Bst DNA polymerase mutant according to claim 5, wherein it is any one of the proteins described in f1-f3 below: f1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1: (1)L68K / I359L / D428K / Y429W; (2)S134P / V143A; (3) S65P / I359L; and, (4)A329D; f2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations, based on the amino acid sequence shown in f1; and, f3: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in f1-f2, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

9. A Bst DNA polymerase mutant, which is any one of the proteins described in g1-g4 below: g1: A protein obtained by mutation based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1, wherein the mutation sites include any one or a combination of at least two of the following: F67, F81, A114, D119, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, S365, Y429, G430, R439, F445, E461, T487, T500, M502, N503, R553, A565, T323, S328, Q348, K468, and E552; g2: A protein with essentially the same enzyme activity and thermal stability obtained by substituting, deleting, or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence shown in g1. g3: A protein that shares over 90% sequence homology with g1, and whose enzyme activity and thermostability are essentially equivalent to those of g1; and, g4: A fusion protein obtained by linking one or more tags or restriction sites to the N-terminus and C-terminus of any of the amino acid sequences described in g1-g3, wherein the tags or restriction sites do not affect the function of the Bst DNA polymerase mutant.

10. The Bst DNA polymerase mutant according to claim 9, wherein the protein in g1 is: The mutation sites include any one or a combination of at least two of the following: F67, A114, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, Y429, G430, R439, F445, E461, T487, M502, N503, R553, A565, T323, S328, Q348, K468, and E552.

11. The Bst DNA polymerase mutant according to claim 9, wherein the mutation comprises any one or a combination of at least two of the following: F67S, F81L, A114L, D119N, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, S365D or I, Y429W, G430E, R439C, F445L or I, E461K, T487N, T500A, M502V, N503S, R553H, A565V, T323A, S328V, Q348L, K468E or N, and E552G.

12. The Bst DNA polymerase mutant according to claim 9, wherein the protein is any one of h1-h3 as described below: h1: A protein obtained by mutation of any combination of the following based on the large fragment of wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID No. 1: (1)V16R / I359L / E461K; (2)S65P / D428K; (3)R133Y / L320P; (4)A193M / N283L; (5) N204R / Q128G; (6) N283L; (7)A409P / N462K; (8)S65P / I359L; (9)L68K; (10)L68V; (11)A114L; (12)Q128G; (13)Q182E / L340R; (14)V281I; (15)V281L; (16)T321A / N503S; (17)S365D; (18)T487N; (19)F445L; (20)F67S / F445L; (21) N204S / S365I; (22)I194V; (23)M33V / F81L / M502V; (24)M502V / R553H; (25)R339C; (26)D377G / R439C; (27)A565V; (28)N204K / T487N; (29)N204R / L320P / F445L; (30)Q128G / N204R / F445I; (31)Q334C; (32)Q334H; (33)G430E; (34)D119N / R339H / I422T; (35)T500A; (36)S134P; (37)V16R / S328V; (38)A329D; (39)E96G / F171S / H278R / Q348L / E552G; (40)L68K / K468E; (41)L68K / K468N; (42)L68K / I359L / D428K / Y429W; (43)I422T; (44)S134P / V143A; and, (45)T323A; h2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations, based on the amino acid sequence shown in h1; and, h3: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in h1-h2, wherein the tags or restriction enzyme sites do not affect the function of the Bst DNA polymerase mutant.

13. The Bst DNA polymerase mutant of claim 9, wherein the protein is any one of the following i1-i3: i1: A protein obtained by any combination of the following mutations based on the wild-type Bst DNA polymerase fragment with the amino acid sequence shown in SEQ ID No. 1: (1) N204R / Q128G; (2)S65P / I359L; (3)F67S / F445L; (4) N204S / S365I; (5)R339C; (6)D377G / R439C; (7) N204K / T487N; (8) N204R / L320P / F445L; (9)Q128G / N204R / F445I; (10)L68K / I359L / D428K / Y429W; (11)S134P / V143A; and, (12)A329D; i2: A protein with substantially the same enzymatic activity and thermostability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations, based on the amino acid sequence shown in i1; and, i3: A fusion protein obtained by linking one or more tags or restriction enzyme sites to the N-terminus and C-terminus of any of the amino acid sequences described in i1-i2, wherein the tag or restriction enzyme site does not affect the function of the Bst DNA polymerase mutant.

14. The Bst DNA polymerase mutant of any one of claims 1-13, wherein, The tag is a His tag, and the enzyme cleavage site is a thrombin cleavage site.

15. The encoding gene of the Bst DNA polymerase mutant according to any one of claims 1-13.

16. The expression vector of the Bst DNA polymerase mutant according to any one of claims 1-13.

17. The expression host bacteria of the Bst DNA polymerase mutant according to any one of claims 1-13.

18. The use of the Bst DNA polymerase mutant according to any one of claims 1-13 in MDA, RCA, LAMP or RT-LAMP.

19. A kit comprising the Bst DNA polymerase mutant according to any one of claims 1-13.

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