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Synthetic pokeweed antiviral protein (PAP) antigen and antibody, and preparation method and application thereof

An artificial synthesis and anti-virus technology, applied in the field of biology and biology, can solve the problems of PAP extraction difficulties, limited content, cumbersome purification steps, etc.

Inactive Publication Date: 2011-12-21
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
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Problems solved by technology

Schlick et al. isolated and purified PAP from Pokeweed plants, and prepared polyclonal antiserum to PAP, which can effectively detect PAP. However, due to the limited content of PAP in Pokeweed (Phytolacca americana) plants, the purification steps are cumbersome and time-consuming. , making the extraction of PAP very difficult (Jean-LucSchlick, Philippe Dulieu, Development of a double sandwich ELISA able to discriminate between free PAP and complexed PAP in leaf extracts. Plant Science 1999, 140: 1-8.)
Our laboratory has used prokaryotic expression products to prepare polyclonal antiserum, but the specificity is poor, which cannot meet the needs of detection of transgenic plants and PAP expression products

Method used

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  • Synthetic pokeweed antiviral protein (PAP) antigen and antibody, and preparation method and application thereof
  • Synthetic pokeweed antiviral protein (PAP) antigen and antibody, and preparation method and application thereof
  • Synthetic pokeweed antiviral protein (PAP) antigen and antibody, and preparation method and application thereof

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Embodiment 1

[0032] 1.1 Design and synthesis of polypeptide sequences

[0033] According to the deduced amino acid sequence (Genbank No.AF338910) (SEQ ID NO3) of the deletion-type PAP gene fragment (deleted N-terminal signal peptide and C-terminal toxic region) transformed by our laboratory, the sequence searched by BLAST and Genbank was carried out. Homologous comparison analysis, using analysis software such as DNAStar and Biosum to predict the secondary structure and epitope of the protein, and selected two epitopes with good immunogenicity and hydrophilicity, a high possibility of linear epitopes, and no predicted epitopes. The polypeptide sequence of the secondary structure, SEQ ID NO1: DISGTERQDVETTLC and SEQ ID NO2: CRYPTLESKAGVKSR. The peptide was synthesized by Shanghai GL peptide Ltd.

[0034] 1.2 Antigen produced by polypeptide coupling

[0035] Since the small molecular polypeptide has no immunogenicity, it needs to be coupled to an immunogenic carrier protein, and the conjug...

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Abstract

The invention relates to a synthetic pokeweed antiviral protein (PAP) antigen and antibody, and a preparation method and application thereof, belonging to the technical field of biology. The synthetic pokeweed antiviral protein antigen comprises a carrier protein with immunogenicity, and a synthetic polypeptide coupled with the carrier protein, wherein the amino acid sequence of the polypeptide is SEQ ID NO 1 or SEQ ID NO 2. The coupler of the synthetic polypeptide and keyhole limpet hemocyanin is used for preparing three PAP antisera; the three PAP antisera can be used for PAP preparation, PAP gene protocaryon expression and detection of eukaryon expression products, transgenic regenerated plants and the like; and the invention can be used for detecting the expression conditions of PAP and gene thereof by immunological means, thereby providing physical foundation for detecting PAP.

Description

technical field [0001] The patent of the present invention belongs to the field of biology and biotechnology, and in particular relates to the technology of preparing pokeweed antiviral protein polyclonal antibody by using artificially synthesized polypeptide as an antigen. Background technique [0002] Pokeweed antiviral protein (PAP) is a single-chain (type I) ribosome inactivating protein (Ribosome Inactivating Proteins, RIPs) isolated from the spring leaves of Pokeweed (Phytolacca americana L.), with a molecular weight of about It is 30kD. RIPs have unique rRNA-N glycosidase activity, which can hydrolyze adenine residues at specific sites on the 28S large subunit RNA GAGA stemloop structure of eukaryotic ribosomes, thereby inactivating ribosomes and interfering with ribosomes Binding to elongation factor, and then inhibiting protein biosynthesis (Irvin, J.D. & Uckun, F.M. Pokeweed antiviral protein: ribosome inactivation and therapeutic applications. Pharmacol. Ther. 19...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/795C07K16/16C07K16/06G01N33/53
Inventor 李莉王锡锋李羽周广和刘艳吴蓓蕾
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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