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Recombination adeno-associated virus of expression human antisense phospho lamban gene and its preparation method

A technology of phospholamban and adenovirus, applied in the field of bioengineering, can solve the problems of immunogenicity of the body, short expression time of adenovirus vector, unsuitability for clinical application, etc., and achieve high titer effect

Inactive Publication Date: 2005-08-24
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression time of adenovirus vector is short, and it is immunogenic to the body, so it is not suitable for future clinical application.

Method used

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  • Recombination adeno-associated virus of expression human antisense phospho lamban gene and its preparation method
  • Recombination adeno-associated virus of expression human antisense phospho lamban gene and its preparation method
  • Recombination adeno-associated virus of expression human antisense phospho lamban gene and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] [Example 1] Cloning of PLB cDNA

[0037] In order to clone human PLB cDNA according to the PLB gene sequence disclosed in Figure 1, PCR primers capable of amplifying PLB cDNA were first designed: the forward primer of 5'-ATTGGATCCGCTGGTATCATGGAG-3 and the reverse primer of 5'-ATCCTCGAGTCAGAGAAGCATCAC-3. RT-PCR was then performed using total RNA extracted from fetal cardiomyocytes as a template. The reverse transcription reaction was carried out in a 20 μl reaction system containing 2 μl of 10× buffer, 4 μl of 25nMMgCl2, 2 μl of 10mmol / L dNTPs, 1 μl of 25U / μl reverse transcriptase (GibcoBRL), and 5 μg extracted from fetal cardiomyocytes by Trizole reagent Total RNA of 50pmol / Lrandom 9mers 1μl, 30°C for 10min, 55°C for 30min, 99°C for 5min, 5°C for 5min, the RT product was used as a PCR template.

[0038] The PCR reaction was carried out in a 50 μl reaction system, 10 μl of the above RT product, 4 μl of 10×PCR buffer, 1 μl of upstream and downstream primers with a final ...

Embodiment 2

[0039] [Example 2] pXXUF 1 · Preparation of asPLB recombinant plasmid

[0040] The PLB gene product obtained in Example 1 was ligated with pXXUF1. The correctly sequenced recombinant pBS·PLB was transduced by the enzyme cutting site, so that both ends of the target gene PLB contained Not I sites, and the restriction endonuclease Not I was used to treat the PLB and the vector pXXUF respectively. 1 Perform agarose gel electrophoresis after single enzyme digestion, the kit recovers the desired fragment, and prepare the recombinant plasmid pXXUF according to the above method 1 · asPLB. PLB cDNA pXXUF 1 In the gfp gene replacement, the plasmid map is shown in Figure 2.

Embodiment 3

[0041] [Example 3] Packaging, recovery and purification of rAAV-asPLB recombinant virus

[0042] 1. Characteristics of natural adeno-associated virus (AAV) and recombinant adeno-associated virus (rAAV) vectors:

[0043] Adeno-associated virus is an animal single-stranded DNA virus belonging to the family Parvoviridae, subfamily Parvoviridae, genus Dependoviridae, naturally defective, non-enveloped and non-pathogenic.

[0044] 1. The AAV genome is a linear, single-stranded (ssDNA) molecule, containing 4680 nucleotides (sequence shown in Figure 1), which is characterized by:

[0045] 1) Its genome consists of four open reading frames (ORFs), which are called rep region, lip region, inf region and cap region (see Figure 1 and Figure 2). A large ORF located at the left end of genomic DNA is called the rep region because frameshift mutations or deletions prevent DNA replication. A large ORF (cap) at the right end codes for the synthesis of three coat proteins. There are two othe...

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Abstract

A recombinant adeno-associated virus (AAV) able to express human antisense phospholamban gene for treating heart failure, cardial infaction and other associated diseases is prepared from natural AAV and adenovirus through artificial shearing, modifying and processing. Its carrier system includes packing plasmid pXX2, auxiliary plasmid pXX6, and eukaryotic expression carriers pXXUF1, and pXXUF3. The coding sequence of human phospholamban gene is inserted in pXXUF1 reversely.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for constructing and preparing a recombinant adeno-associated virus (rAAV-asPLB) capable of expressing human antisense phospholamban gene, and more specifically relates to the cloning of human phospholamban gene and its antisense A method for packaging and preparing the recombinant adeno-associated virus recombinant of the phosphoprotein gene, and the pharmaceutical application of the recombinant adeno-associated virus recombinant. Background technique [0002] Phospholamban (PLB for short) is a protein with a small molecular weight, consisting of 52 amino acid residues, mainly present in cardiac muscle and smooth muscle (J Biol Chem, 249: 6174-6180 (1974)). In vitro studies suggest that PLB can be phosphorylated by different protein kinases, and phosphorylated PLB relieves its inhibitory effect on SERCA2a, causing phosphorylation-mediated stimulation (J Biol Chem, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N7/01C12N15/66C12N15/861
Inventor 汪道文肖啸赵春霞汪培华
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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