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Adeno-associated virus mutant and application thereof
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A technology of mutants and virions, applied in the directions of viruses/phages, viruses, applications, etc., to achieve a wide range of applications, good infectivity, and good targeting.
Active Publication Date: 2021-12-07
GUANGZHOU PACKGENE BIOTECH CO LTD
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[0006] Chinese patent document CN106062200B discloses a recombinant adeno-associated virus vector, including a variant AAV2 capsid protein, the variant AAV2 capsid protein contains at least 4 amino acid substitutions relative to the wild-type AAV2 capsid protein, and its recombinant adeno The use of related viral vectors in the preparation of drugs for delivering gene products to the neurons, retina or liver tissue of subjects; currently, there is still little research on the effect of recombinant adeno-associated viruses on the heart, and there is still a need to improve recombinant adeno-associated viruses Characterization of in vivo transgene expression and tissue specificity
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Embodiment 1
[0039] Example 1, construction of AAV9 capsid protein mutant library backbone vector and mutant Rep-CAP vector
[0040] 1. Construction of AAV9 capsid protein mutant library backbone plasmid:
[0041]The backbone vector contains the AAV5 p41 promoter fragment, the AAV2 rep splicing signal sequence, and the CAP sequence of the mutated frameshifted AAV9 (wherein the CAP sequence of AAV9 mutates the K at the original amino acid position 449 to R, and the nucleic acid sequence is mutated from tcaaag to tctaga, Introduce the XbaI restriction site; mutate the nucleic acid sequence of amino acid G at the original amino acid position 594 from ggc to ggt, and introduce the BshTI restriction site; at the same time, insert a stop codon between the original amino acid position 588 and position 589 34bp sequence to cause a frame shift of the sequence and avoid contamination caused by unclean digestion of the backbone vector), and add the SV40 polyA sequence after the CAP sequence. The abo...
Embodiment 2
[0055] Embodiment 2, construction AAV capsid protein mutant
[0056] 1. Construction of mutant serotype vectors:
[0057] 1) Digest the backbone: Digest the Rep-CAP plasmid of AAV9 with BsiWI and AfeI, perform gel electrophoresis and cut off the band of the desired fragment size for gel recovery;
[0058] 2) Target fragment: the target plasmid AAV capsid protein mutant obtained by screening was double digested with BsiWI and AfeI, gel electrophoresis, and the band of the target fragment size was cut out for gel recovery;
[0059] 3) Connection: Take a clean 200uL PCR tube, mark it and put it on the ice box, prepare the reaction solution with the above enzyme-cut backbone and the target fragment at a molar ratio of 1:3, and react in a PCR instrument for 30 minutes at 16°C for connection;
[0060] 4) Transformation: Thaw 50 μL of competent cells on ice; mix 10 μL of the ligation product with stbl3 competent cells, and place on ice for 20-30 minutes; heat shock at 42°C for 45 se...
Embodiment 3
[0066] Embodiment 3, the influence of AAV capsid protein mutant on mouse heart
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Abstract
The invention relates to a virosome in the biological field and application thereof, and particularly discloses an adeno-associated virus mutant with heart targeting and application thereof. A heterologous peptide is (a) a protein consisting of an amino acid sequence shown as SEQ ID No: 14, or (b) a protein which is derived from the (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence in the (a), and has heterologous peptide activity. A recombinant adeno-associated virus virosome constructed by utilizing an AAV capsid protein mutant has high specificity, high safety, low immunogenicity, wide application range, high heart infectivity, high targeting property and long time for expressing an exogenous gene in a mouse.
Description
[0001] This application is a divisional application of CN112194706A (application date is September 30, 2020, application number is 202011070091.8, and the invention name is adeno-associated virus mutant and its application). technical field [0002] The invention relates to virosomes in the biological field and applications thereof, in particular to a cardiac-targeted adeno-associated virus mutant and applications thereof. Background technique [0003] Adeno-associated virus (adeno-associated virus, AAV), also known as adeno-associated virus, belongs to the family Parvoviridae and is the most simple single-stranded DNA-deficient virus found so far. It needs helper virus (usually adenovirus) virus) involved in replication. The AAV genome is a single-stranded DNA fragment of about 4.7kb, contained in a 20nm-diameter regular icosahedral non-enveloped viral capsid, and can be divided into three functional regions: two open reading frames (Rep gene, Cap gene) and the terminal In...
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