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Anti-human ppGa1NAc-T2 monoclonal antibody and application thereof

A monoclonal antibody and unit technology, applied in the field of microbial engineering, can solve the problems of high late gene expression and regulation mechanism of the virus are not very clear, weak research on baculovirus genomics, and immature multiple expression technology, etc., to achieve high affinity, Reduced requirements, highly specific effects

Inactive Publication Date: 2012-10-03
SUZHOU UNIV
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Problems solved by technology

[0006] However, there are deficiencies in their plan: First, they used the insect baculovirus expression system (Sf9 cells) to prepare the immunogen, but there are some problems such as the relatively weak genomics research of baculovirus, and the knowledge about the late gene of the virus. The mechanism of high expression and regulation is still not very clear, the purification of expression products is relatively difficult, and the technology of multiple expression is not mature enough, etc., all of which need to be further solved; secondly, the prepared antibodies have good performance in ELISA and immunoprecipitation. However, the performance in Western Blot experiments is not satisfactory, which limits the use of this antibody, because Western blotting is now the most general and reliable means of quantitatively detecting the expression of the target protein

Method used

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  • Anti-human ppGa1NAc-T2 monoclonal antibody and application thereof
  • Anti-human ppGa1NAc-T2 monoclonal antibody and application thereof
  • Anti-human ppGa1NAc-T2 monoclonal antibody and application thereof

Examples

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Embodiment 1

[0044] Example 1: Preparation of mouse anti-human ppGalNAc-T2 protein monoclonal antibody

[0045] The strategy of using the prokaryotic expression product of ppGalNAc-T2 gene as the immunogen to immunize Balb / C mice to obtain the B cells sensitized by the immunogen, the specific operation method:

[0046] 1. Preparation of Monoclonal Antibody Immunogen

[0047] 1.1 Amplification of the complete open reading frame fragment of 1ppGalNAc-T2 gene

[0048] It is very difficult to obtain purified ppGalNAc-T2 protein directly from tissues or cells to prepare antibodies. Therefore, it is an effective method to prepare antibodies through genetic engineering expression products, and obtaining genetic engineering expression products to transfer target genes is a prerequisite.

[0049] The cell line 293T can produce a large number of ppGalNAc-T2 gene transcripts, and then extract the total RNA, RT-PCR amplification and construct the recombinant transfer vector pGEM-T-ppGalNAc-T2, and se...

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Abstract

The invention belongs to the field of microbiology engineering, in particular relating to an anti-human ppGa1Nac-T2 monoclonal antibody as well as preparation method and application thereof. The monoclonal antibody is generated by a hybridoma cell strain LW-5F3. By utilizing the anti-human ppGa1Nac-T2 monoclonal antibody with high specificity and high titer, rapid flux detection can be carried out on expression of ppGa1Nac-T2 through various approaches such as enzyme-linked immunosorbent assay (ELISA), Western-blot, Immunocytoflurescence, Flowcytometer analysis or Immunohistochemistry and thelike.

Description

technical field [0001] The invention belongs to the field of microbial engineering, and in particular relates to an anti-human ppGalNAc-T2 monoclonal antibody and its preparation method and application. Background technique [0002] Polypeptide: The N-acetylgalactosaminyl transferase (ppGalNAc-T) family is the initial enzyme that catalyzes the serine or threonine (Ser / Thr) residues on the protein peptide chain to synthesize O-sugar chains. So far, at least 18 members of this enzyme family have been cloned and expressed in mammals. The comparative analysis of gene databases suggested that the family consisted of 24 potential members. All ppGalNAc-Ts can bind to the substrate UDP-GalNAc, but they have different catalytic sites on the protein backbone, which makes the enzyme a large number of members and has a wide tissue distribution. Recent studies have shown that ppGalNAc-T family members are abnormally expressed in a variety of human tumor cells or tissues, for example, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577
Inventor 刘春亮林丹丹顾文超姜智徐岚吴士良
Owner SUZHOU UNIV
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