ppgalnac-t20 antigen and preparation method of polyclonal antibody thereof
A ppgalnac-t20, polyclonal antibody technology, applied in the field of antibody and its preparation, the preparation of ppGalNAc-T20 antigen and its polyclonal antibody, can solve the problems of high price, poor immunogenicity, etc., achieve easy purification, overcome the immunogenicity poor sex effect
Inactive Publication Date: 2011-09-28
SHANGHAI JIAOTONG UNIV
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Problems solved by technology
The antibody prepared by the present invention does not cross-react with ppGalNAc-T10, which has the highest homology with ppGalNAc-T20, and does not cross-react with other members of the ppGalNAc-Ts family; the present invention prepares the required antibody through simple molecular cloning experiments. Antigenic protein, and the antigenic protein and GST fusion expression, easy to purify; the present invention overcomes the disadvantages of poor immunogenicity and high price of synthetic polypeptide methods
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[0034] Preparation of ppGalNAc-T20 polyclonal antibody
[0035] Step 1, establishment of antigen sequence
[0036] Input the protein sequences of ppGalNAc-T20 (GenBank Accession Number: GU220060) and ppGalNAc-T10 (GenBank Accession Number: AJ505950) into the ClustalX software for comparison, and select a sequence short T20 in the stem region of the ppGalNAc-T20 protein as the antigen, short The sequence of T20 is shown as SEQ ID NO: 2 (ppGalNAc-T20 full protein codes from 30AA to 141AA);
[0037] Step 2, construction of recombinant plasmid
[0038] The DNA sequence corresponding to the amino acid sequence of the antigen short T20 is shown in SEQ ID NO: 1; primers were designed according to the multiple cloning site of pGEX-5X-1 and the DNA sequence of short T20:
[0039] Upstream primer: 5'-GTT GCG AGC GGC CGC CTG TAC AAG GAT-3'; (SEQ ID NO: 3);
[0040] Downstream primer: 5'-GATAAT GAG TCG ACC GTT TGG CAG CCTTTC-3' (SEQ ID NO: 4).
[0041] Using ppGalNAc-T20 full-length cDN...
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The present invention relates to a ppGalNAc-T20 antigen and a preparation method of a polyclonal antibody thereof. An amino acid sequence of the ppGalNAc-T20 antigen is shown as SEQ ID NO: 2. The invention also relates to separated nucleic acid, a base sequence of which is shown as SEQ ID NO: 1, and also relates to a polyclonal antibody which is prepared by adopting the following steps of: taking proteinshown as the amino acid sequence SEQ ID NO: 2 as an antigen, and preparing antiserum by adopting a conventional polyclonal antibody preparation method; and purifying the antiserum to obtain the polyclonal antibody. The invention also relates to a method for preparing the polyclonal antibody, comprising the following steps of: cloning nucleic acid fragments shown as the SEQ ID NO: 1; establishing recombinant plasmids, converting colibacillus, culturing, inducing, pyrolyzing and purifying to obtain protein; taking the protein as the antigen, preparing the antiserum by adopting the conventional polyclonal antibody preparation method, and purifying the antiserum to obtain the polyclonal antibody. The ppGalNAc-T20 antigen and the preparation method of a polyclonal antibody thereof provided by the present invention have simple method, and the prepared antibody has strong immunogenicity.
Description
technical field [0001] The invention relates to an antibody in the field of biotechnology and a preparation method thereof, in particular to a preparation method of a ppGalNAc-T20 antigen and a polyclonal antibody thereof. Background technique [0002] UDP-galactosamide: N-acetylgalactosaminyl transferase (ppGalNAc-Ts) is the first step of the O-type glycoprotein sugar chain synthesis reaction to initiate glycosyltransferase, which can convert N-acetylgalactosaminyl Transferred to serine or threonine residues in the protein peptide chain. Nineteen members of the ppGalNAc-Ts family in humans have been reported and studied, and their distribution in human tissues and the specificity of glycosylation modification of polypeptide substrates in vitro all have different degrees of difference. ppGalNAc-Ts can be used as a tumor marker, such as ppGalNAc-T13, which is specifically highly expressed in neuroblastoma-derived cell lines, and can be used as an early diagnostic marker for ...
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IPC IPC(8): C12N9/10C12N15/54C12N15/70C07K16/40C07K16/06
Inventor 张延彭灿武功冬邹霞谢文娴
Owner SHANGHAI JIAOTONG UNIV