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Recombinant adeno-associated virus particle and its application

A technology of virus particles and viruses, applied in the field of genetic engineering, can solve the problem of large doses, and achieve the effect of reducing damage and strong infection ability

Active Publication Date: 2020-12-01
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, relatively speaking, the dose of AAV6 reported in the literature is relatively large, usually (6~9)×10 9 The total amount of virus particles in vg can achieve effective fertilized egg infection

Method used

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  • Recombinant adeno-associated virus particle and its application
  • Recombinant adeno-associated virus particle and its application
  • Recombinant adeno-associated virus particle and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] In the present embodiment, the inventors are screened by constructing a random mutant library of a AAV6 peptide, and is screened to a new AAV6 mutant inserted into seven amino acids.

[0050] Library preparation

[0051] 1.1 Chemical Synthesis AAV6-7MER-NNS below two segments:

[0052] 5'cctccagagcagcagcnnsnnsnnsnnsnnsnnsnnsnnsacagaccctgcg 3 ';

[0053] 5'cggtcgcagggtctgtsnnsnnsnnsnnsnnsnnsnnngctgctgctctG 3 ';

[0054] Where NNS represents a random coding sequence.

[0055] 1.2 The synthetic AAV6-7MER-NNS positive chain and the reverse link were added 10 μl (primer final concentration of 10 mm) using annealing to obtain an AAV6-7MER-NNS template. The annealing program is: 95 ° C, 5 min; 95 ° C, 1 min; 92 min, 1 min; 4 ° C, 60 min. Among them, the second step and third steps, each cycle drops 3 ° C, a total of 25 cycles.

[0056] 1.3 Plasmid PAAV-SHORT UBC-MSCARLET-POLYA-P40-AAV6-CAP-FLEX-SV40 POLYA (its structure and insertion site figure 1 The Single enzyme cut is performe...

Embodiment 2

[0088] Example 2 AAV6 mutant NS01 construction and virus packaging

[0089] 1. AAV6-NS01-DNA fragment Cagacgacggacaagtacaag is inserted at 588-589 for the amino acid at 588-589 to obtain serotome carrier AAV6-NS01.

[0090] 2. Using shuttle carrier PAAV-CMV-EGFP-WPRE-POLYA (vector map Figure 2A AAV virus is packaged as a serotype carrier as a serotype carrier in AAV6-NS01.

[0091] 3. Titness assays were measured using WPRE primers. And use the inspection confirmation VP1, VP2, and VP3 expression is normal.

Embodiment 3

[0092] Example 3 AAV6 mutant NS01 infection of affected egg

[0093] Get affected egg

[0094] The wild-type C57BL / 6J mice in this embodiment are the products of Shanghai Squel-Bikai Experimental Animal Co., Ltd. Embryonic operation fluid M2 and embryonic culture solution M16, ovate cell group digestion liquid hyaluronic acid enzyme HYALURONIDase, embryo-covered mineral oil is used to maintain the stability of osmosis in culture droplets, and the above products are from Sigma, product The catalog numbers are M7167, M7292, H4272, M8410, respectively.

[0095] In order to increase the fertilized eggs operable in the experiment, multi-eggs induced by human injection hormones. PMSG (pregnancy vessel serum) is an analog FSH (follicle stimulatory) to promote follicle growth and development, HCG (human chorionic gonadotropin) is an simulated LH (yellow body) promoting ovulation effect, here two hormones From Ningbo Second Hormone Factory.

[0096] Mice affailed eggs from 4-6W C57BL / 6...

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Abstract

The invention relates to the field of genetic engineering, in particular to a recombinant adeno-associated virus particle and its application. The recombinant adeno-associated virus particle comprises a mutated AAV6 capsid protein, the mutated adeno-associated virus type 6 capsid protein, wherein relative to the corresponding wild-type AAV6 capsid protein, the amino acid fragment QTTDKYK is inserted into the mutated AAV6 capsid protein , the insertion site is between amino acids 588 and 589 of the wild-type AAV6 capsid protein. The recombinant adeno-associated virus particle has stronger infectivity, and thus can achieve substantially the same infective effect as a common dose at a lower dosage, thereby effectively reducing damage to host cells during the transduction process.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, and in particular, there is a recombinant adrenal associated viral particle and its application thereof. Background technique [0002] The research of human diseases in human diseases using a transgenic disease model has been used, more than just provide an important fundamental basis for research on the pathogenesis of the disease, but also avoids the risk and ethical issues caused by human experiments. The construction of transgenic animal disease models has promoted human understanding and research on genetic functions while promoting clinical experience accumulation and drug development of corresponding rare diseases. [0003] The construction of transgenic mice is to use CRISPR-Cas9 gene editing techniques, which is very convenient, but relying on high prices, performing fine microscopic equipment and operators who have trained experience. At present, the research on the presence of the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/015C12N15/864C12N5/10A61K48/00
CPCA61K48/0008A61K48/005C07K14/005C12N15/86C12N2750/14122C12N2750/14123C12N2750/14144
Inventor 杨兴林贾国栋杨佳丽李琴夏清梅
Owner OBIO TECH SHANGHAI CORP LTD
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