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Polyclonal antibody for specifically recognizing AAV9 capsid protein and preparation method thereof

A polyclonal antibody, capsid protein technology, applied in the field of medicine and biochemical industry, can solve the problem of lack of specificity of antibodies

Active Publication Date: 2022-07-01
CHINA THREE GORGES UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Due to the high conservation among different serotypes of AAV, most AAV capsid protein antibodies lack specificity, and often one antibody recognizes multiple serotypes of AAV. Currently, there is no commercialization specific for AAV9 capsid protein on the market Antibody

Method used

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  • Polyclonal antibody for specifically recognizing AAV9 capsid protein and preparation method thereof
  • Polyclonal antibody for specifically recognizing AAV9 capsid protein and preparation method thereof
  • Polyclonal antibody for specifically recognizing AAV9 capsid protein and preparation method thereof

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Embodiment 1

[0026] AAV capsid variable region protein sequence was obtained

[0027] The amino acid sequence of AAV1-AAV10 capsid protein was downloaded from the NCBI website, imported into the software DNAMAN, sequence homology alignment analysis, and found 8 AAV9 variable regions (Aa262-269, Aa448-483, Aa488-510, Aa527- 540, Aa545-557, Aa576-601, Aa661-668, Aa706-718), splicing the variable region sequence, the obtained sequence is the AAV9 capsid protein variable region sequence, and the DNA sequence encoding the variable region is composed of gold. The pET-30a prokaryotic expression vector was synthesized and constructed by Weizhi Biotechnology Co., Ltd. The 5'-end restriction site was XhoI, and the 3'-end restriction site was NdeI. The AAV9 variable region plasmid pET30a-AAV9-VR was digested with XhoI+NdeI and separated by 1% agarose gel electrophoresis. Two restriction fragments of 5500bp and 1000bp were found (Figure 1). The results were consistent with expectations. The recombina...

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Abstract

The invention provides a polyclonal antibody capable of specifically recognizing AAV9 capsid protein and a preparation method thereof, eight AAV9 capsid variable region amino acid sequences are sequentially connected to obtain an amino acid sequence SEQ ID NO.1 of AAV9 variable region VR, a DNA sequence such as SEQ ID NO.2 for coding the AAV9 capsid variable region is obtained from the amino acid sequence, the DNA sequence is inserted into a prokaryotic expression vector pET30a to obtain a plasmid pET30a-AAV-VR, and the plasmid pET30a-AAV-VR is subjected to expression by virtue of a prokaryotic expression vector pET30a to obtain the polyclonal antibody capable of specifically recognizing AAV9 capsid protein. The invention relates to application of an AAV9 variable region protein immune Japanese white rabbit in preparing a polyclonal antibody, detecting antibody titer by enzyme-linked immunosorbent assay (ELISA), Western-blot and cell immunofluorescence detection antibody, and particularly relates to application of the AAV9 variable region protein immune Japanese white rabbit in preparing the polyclonal antibody, detecting antibody titer by enzyme-linked immunosorbent assay (ELISA), and detecting antibody titer by using a cell immunofluorescence method. The titer of the antibody is 1: 10240000, and the antibody can specifically recognize an AAV9 capsid protein sequence. And a foundation is laid for subsequent AAV vector development and AAV biological function research.

Description

technical field [0001] The invention relates to the technical field of medicine, biochemical industry, in particular to a method for preparing a polyclonal antibody that specifically recognizes AAV9 capsid protein. Background technique [0002] Adeno-associated virus (AAV) belongs to the genus Dependovirus of the family Parvoviridae. The virus exists widely in vertebrates such as humans and primates. At present, the scientific community generally believes that AAV does not cause human diseases. In recent years, the number of clinical trials using AAV vectors for in vivo gene therapy by domestic and foreign scientific research platforms has gradually increased. Its excellent safety and efficient transduction of targeted organs and tissues make it the preferred vector system for in vivo gene therapy. . AAV virions are about 25 nm in diameter and encapsulate a 4.7 kb single-stranded DNA genome. The genome consists of Rep and Cap genes, flanked by inverted terminal repeats (ITR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C07K16/06C12N15/70C12N15/35G01N33/569
CPCC07K16/081C07K16/065C12N15/70C07K14/005G01N33/56983C07K2317/56C12N2750/14122G01N2333/015Y02A50/30
Inventor 吕亚丰曹春雨李舒月张浩秦宇杨建林王静
Owner CHINA THREE GORGES UNIV
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