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Vacpox virus capping enzyme mutant, recombinant vector, recombinant engineering bacterium and application of vaccpox virus capping enzyme mutant, recombinant vector and recombinant engineering bacterium

A vaccinia virus, recombinant vector technology, applied in the direction of virus/phage, vector, nucleic acid vector, etc., can solve the problems of poor thermal stability and difficult to meet actual needs, etc.

Pending Publication Date: 2022-04-19
苏州瀚源新酶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the vaccinia virus capping enzyme mutants on the market have poor thermal stability, which is difficult to meet the actual needs

Method used

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  • Vacpox virus capping enzyme mutant, recombinant vector, recombinant engineering bacterium and application of vaccpox virus capping enzyme mutant, recombinant vector and recombinant engineering bacterium
  • Vacpox virus capping enzyme mutant, recombinant vector, recombinant engineering bacterium and application of vaccpox virus capping enzyme mutant, recombinant vector and recombinant engineering bacterium
  • Vacpox virus capping enzyme mutant, recombinant vector, recombinant engineering bacterium and application of vaccpox virus capping enzyme mutant, recombinant vector and recombinant engineering bacterium

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Experimental program
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Effect test

preparation example Construction

[0051] One embodiment of the present invention provides a method for preparing the above-mentioned recombinant vector, which includes the following steps: using a first amplification primer pair to perform PCR amplification on the first vector to obtain a recombinant vector, wherein the first vector contains the recombinant vector shown in SEQ ID No.2. The coding sequence corresponding to the indicated amino acid sequence. The first amplification primer pair contains the nucleotide sequence corresponding to the mutation site of the vaccinia virus capping enzyme mutant.

[0052] Wherein, the first amplification primer pair is selected from at least one pair of the following primer pairs: a primer pair with a nucleotide sequence such as SEQ ID No.20 and SEQ ID No.21, a nucleotide sequence such as SEQ ID No.22 and the primer pair shown in SEQ ID No.23, the primer pair shown in SEQ ID No.24 and SEQ ID No.25, the nucleotide sequence shown in SEQ ID No.26 and SEQ ID No.27 The prime...

Embodiment 1

[0067] 1. Cloning of wild-type vaccinia virus capping enzyme gene

[0068] The wild-type vaccinia virus capping enzyme gene was codon-optimized with Escherichia coli as the host cell, and the optimized vaccinia virus capping enzyme gene was obtained, named VCE, its nucleotide sequence was SEQ ID No.1, and the expressed amino acid sequence It is SEQ ID No.2.

[0069]Taking SEQ ID No.1 as the target gene, the upstream amplification primer SEQ ID No.18 and the downstream amplification primer SEQ ID No.19 were used to amplify the target gene. Amplification conditions were as follows: amplification at 95°C for 2 min, then amplification at 56°C for 20 sec, amplification at 72°C for 90 sec, a total of 30 cycles, and finally amplification at 72°C for 10 min.

[0070] After the reaction was completed, the PCR amplified product was detected by 1.5% agarose gel electrophoresis, and a 1.0 kb band was obtained, the length of which was in line with the expected result. According to the st...

Embodiment 2

[0074] Construction, expression and purification of capping enzyme mutants of vaccinia virus

[0075] This application simulates the protein analog crystal structure of the wild-type vaccinia virus capping enzyme, analyzes and designs mutation sites according to the protein analog crystal structure, and finds the wild-type vaccinia virus capping enzyme whose amino acid sequence is shown in SEQ ID No.2 Amino acids at positions 424, 461, 513, 812, and 750 have a greater impact on the thermal stability of the enzyme, and the amino acid sequence V424I as shown in SEQ ID No.2 was discovered and verified at the same time The thermostability of the vaccinia virus capping enzyme mutant obtained by at least one of mutation, Y461F mutation, I513V mutation and V750L mutation is relatively high. The schematic diagram of the protein analog crystal structure of the wild-type vaccinia virus capping enzyme is as follows figure 1 shown.

[0076] 1. Construction of single point mutants of VCE...

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Abstract

The invention relates to a vaccinia virus capping enzyme mutant, a recombinant vector, a recombinant engineering bacterium and application of the vaccinia virus capping enzyme mutant. The vaccinia virus capping enzyme mutant comprises: (a) a polypeptide formed by deleting, replacing or adding one or more amino acids to an amino acid sequence as shown in SEQ ID No.2; or (b) a polypeptide having at least 90% of homology with the polypeptide composed of the amino acid sequence as shown in SEQ ID No.2. The half-life period of the vaccinia virus capping enzyme mutant at 45 DEG C is 35 min or above and is higher than that of a wild type vaccinia virus capping enzyme mutant, and when mRNA is capped at a high temperature, the vaccinia virus capping enzyme mutant still shows excellent catalytic activity and can play an important role in the fields of clinical medicine, influenza vaccines, tumor vaccines, biological pharmacy and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a vaccinia virus capping enzyme mutant, a recombinant vector, a recombinant engineering bacterium and applications thereof. Background technique [0002] In eukaryotes, mRNA is post-transcriptionally modified to form a special structure at the 5' end, namely the cap structure, which plays an important role in the stability, transport and translation of mRNA. Vaccinia virus capping enzyme is an effective enzyme that catalyzes the formation of a cap structure. It consists of two subunits, D1 and D12, and has RNA triphosphatase activity, guanylyltransferase activity and guanine methyltransferase activity. A 7-methylguanine cap (m7Gppp) was ligated to the 5' end of the RNA (m7Gppp5'N). Vaccinia virus capping enzyme, in the presence of appropriate concentrations of capping buffer (Capping buffer), guanosine triphosphate (GTP), S-adenosylmethionine (SAM), etc., can be used within one hour...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N9/10C12N15/70C12N1/21C12P19/34C12R1/19
CPCC12N9/16C12N9/1007C12N9/1029C12N15/70C12P19/34C12N2800/101C12N2800/22
Inventor 马富强罗成坤陆泽林王志耘杨广宇
Owner 苏州瀚源新酶生物科技有限公司
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