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CN-122012454-A - Phi29 DNA polymerase variant and application thereof

CN122012454ACN 122012454 ACN122012454 ACN 122012454ACN-122012454-A

Abstract

The application provides a Phi29 DNA polymerase variant and the application thereof. Compared to wild-type enzymes, the Phi29 DNA polymerase variant comprising A484N+S395T, A484Q+S395T, A484Q+T534C, Y281H+S395T, D186E+G401K or S395T+K402R substitution groups described in this application exhibits higher DNA synthesis fidelity, effectively reduces errors during amplification, thereby reducing the risk of non-specific amplification, and has good industrial application prospects.

Inventors

  • Jin Qiuheng
  • HE WEI
  • WU ZHI
  • LIU MINGJUN

Assignees

  • 南京诺唯赞生物科技股份有限公司

Dates

Publication Date
20260512
Application Date
20260415

Claims (9)

  1. Phi29 DNA polymerase variant, characterized in that the amino acid sequence of the variant comprises compared to SEQ ID NO. 1 any substitution group selected from the group consisting of A484N+S395T, A Q+S395T, A Q+T534C, Y281H+S395T, D E+G401K or S395T+K402R, the amino acid sequence of the variant being as shown in any one of SEQ ID NO. 7-12.
  2. 2. A biomaterial, characterized by one or more selected from the group consisting of: a) A polynucleotide molecule encoding a Phi29 DNA polymerase variant of claim 1; b) An expression vector comprising a polynucleotide molecule as described in a); c) A host cell comprising a polynucleotide molecule as described in a), or a host cell comprising an expression vector as described in b).
  3. 3. The biomaterial according to claim 2, wherein the polynucleotide molecule has a nucleotide sequence as set forth in any one of SEQ ID NOs 19 to 24.
  4. 4. A method for producing the variant according to claim 1, comprising (1) culturing the host cell in the biological material according to claim 2, and (2) recovering the Phi29 DNA polymerase variant.
  5. 5. A composition comprising the Phi29 DNA polymerase variant of claim 1.
  6. 6. A kit comprising the Phi29 DNA polymerase variant of claim 1.
  7. 7. Use of the Phi29 DNA polymerase variant of claim 1 for in vitro synthesis of DNA.
  8. 8. A method for preparing DNA, comprising contacting a DNA template, primer, modified or unmodified nucleoside triphosphate with the Phi29 DNA polymerase variant of claim 1, and incubating in a reaction system to obtain a target DNA.
  9. 9. Use of a Phi29 DNA polymerase variant of claim 1 in a DNA preparation method that improves the fidelity of the product DNA.

Description

Phi29 DNA polymerase variant and application thereof Technical Field The application belongs to the technical field of biology, and particularly relates to a Phi29 DNA polymerase variant and application thereof. Background DNA vaccines have proven to be one of the most promising applications in the field of gene therapy. Because of its unique ability to easily induce humoral and cellular immune responses, this concept, when first demonstrated in the early 1990 s, has attracted much attention to （Tang, D. C. et., al. (1992). Genetic immunization is a simple method for eliciting an immune response. Nature, 356(6365), 152–154;Ulmer, J. B. et., al. (1993). Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 259(5102), 1745–1749; Fynan, E. F., et., al. (1993). DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations）. by vaccine developers traditional methods of DNA vaccine preparation based on intracellular replication, at high cost, and the need to avoid contamination of cultures, especially when using the universal escherichia coli (ESCHERICHIA COLI) expression system, requires complex downstream purification processes to remove bacterial endotoxins toxic to mammals, greatly increasing production costs. The Touchlight company in the united kingdom has proposed doggyboneDNA (dbDNA) vector, and has not used the traditional method of preparing plasmid DNA by intracellular replication, but has used in vitro enzymatic synthesis of DNA. dbDNA is a small, linear double-stranded DNA structure shaped like a "dog bone". The synthesis mainly comprises two steps of 1) DNA amplification, wherein an initial template of the DNA is circular plasmid DNA, and the initial template comprises a target gene sequence, a Phi29 DNA polymerase recognition sequence (telRL) derived from ESCHERICHIA PHAGE N and a bacterial skeleton sequence. Converting the DNA into two single-stranded circular DNA by denaturation treatment, performing rolling circle amplification by using polymerase from Bacillus subtilis phage phi29 to generate long linear double-stranded concatemer DNA with telomerase recognition sequence interval, and 2) cutting and covalently closing TelN telomerase, wherein the TelN telomerase can bind to the telomerase recognition site to recognize telRL sites on two sides of a target gene expression element, and cutting and connecting to generate linear gene doggyboneDNA with covalently connected tail ends. Under the current conditions, phi29 DNA polymerase reduces the generation of wrong paired products through 3'-5' exo-activity in the process of preparing DNA products, and wrong substrates can be introduced in the process of preparing DNA products, so that impurities （Del Prado, A. et., al. (2019). New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 dna polymerase. Scientific Reports, 9(1) ）. are generated, and therefore, a Phi29 DNA polymerase which is suitable for economical use and has high fidelity is needed. Disclosure of Invention In a first aspect, the application provides a Phi29 DNA polymerase variant comprising an amino acid sequence comprising at least one mutation selected from the group consisting of A484, Q497, S395, M8, T534, G401, Y281, D186, K402 compared to SEQ ID NO.1, wherein said mutation is of the type selected from the group consisting of substitution or deletion. In a second aspect, the present application provides a class of biological materials selected from one or more of the following: a) A polynucleotide molecule encoding the variant; b) An expression vector comprising a polynucleotide molecule as described in a); c) A host cell comprising a polynucleotide molecule as described in a), or a host cell comprising an expression vector as described in b). In a third aspect, the present application provides a method of preparing the Phi29 DNA polymerase variant described above. In a fourth aspect, the application provides a composition comprising at least one Phi29 DNA polymerase variant according to the application. In a fifth aspect, the application provides a kit comprising at least one Phi29 DNA polymerase variant according to the application. In a sixth aspect, the application provides the use of a Phi29 DNA polymerase variant, composition or kit as described above in the preparation of DNA. Detailed Description Phi29 DNA polymerase variants The present application provides a Phi29 DNA polymerase variant having an amino acid sequence which has at least 95% sequence identity compared to SEQ ID NO. 1 and comprising at least one mutation selected from the group consisting of A484, Q497, S395, M8, T534, G401, Y281, D186, K402, wherein said mutation type is selected from the group consisting of substitution or deletion, relative to SEQ ID NO. 1. In some embodiments, the mutation type is a substitution. In some embodiments, the substitution at position a484 is Q or N. In some embod