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
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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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