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Single-chain fragment antibody-polypeptide amalgamation protein and uses thereof

A technology of fusion proteins and fragments, applied in the fields of biology and medicine, can solve the problems of weak gene expression effect and cannot meet clinical application, etc., and achieve the effect of improving expression

Active Publication Date: 2008-09-24
GUANGZHOU ANJIE BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the weak effect of these traditional antisense gene methods on inhibiting gene expression, they cannot meet the needs of clinical application.

Method used

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  • Single-chain fragment antibody-polypeptide amalgamation protein and uses thereof
  • Single-chain fragment antibody-polypeptide amalgamation protein and uses thereof
  • Single-chain fragment antibody-polypeptide amalgamation protein and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038]Example 1. Construction of a plasmid with a fusion protein that binds to Her2 receptor and carries a bidirectional function of siRNA drug. After finding the full-length sequence of the Her2 single-chain fragment antibody from the gene library, use primer software to design relevant primers.

[0039] Designed Her2-ScFv primers:

[0040] Forward primer: NcoI is an endonuclease

[0041] 5'-GGGCCATGGCCCAGGTGCAGCTGTTGCAGTCTGGGGCAGAG-3'

[0042] Anti-primer: NotI is an endonuclease

[0043] 5'-TTGCGGCCGCTCCGGAATTCACCTAGGACGGTCAGCTTGGTCCC-3'

[0044] Designed protidine peptide primers:

[0045] Positive primer: NotI is an endonuclease

[0046] 5'-CCGGAGCGGGCCGCAATGGCCAGGTACAGATGCTG-3'

[0047] Anti-primer: PpuMI as endonuclease, stop codon and 6 histidines

[0048] 5'-GCCGGGTCCCAGGAAAGGATCAGATCTGCATTAATGGTGGTGGTGATGATGAGATCTGTGTCTTTCTACATCTCGGTCTG-3'

[0049] Clone relevant target fragments by PCR: Her2-ScFv PCR reaction system 1:50ul system:

[0050] wxya 2 O 37ul

...

Embodiment 2

[0100] Example 2 Small amount extraction of pACgp67B-Her2-scfv-protamine plasmid

[0101]Collect the bacteria by centrifugation at 12000g×1min. (Generally, 3-4ml bacterial solution is used for extracting a portion), discard the supernatant. Add 250ul ice-bath Buffer S1 (containing RNaseA), vortex and oscillate to fully suspend the bacteria. Add 250ul Buffer S2 and mix gently 6 times. (This process does not exceed 5 minutes) Add 350ul Buffer S3, and mix gently 6 times. Centrifuge at 12000g x 10min. Take the supernatant, pass through the DNA preparation tube at 12000g×1min, and discard the filtrate. Add 700ul BufferW1 to the DNA preparation tube, 12000g×1min, discard the filtrate. Add 500ul Buffer W2 to the DNA preparation tube, 12000g×1min, discard the filtrate. Centrifuge again at 12000 g x 1 min. Add 40ul of ddH2O (or EB buffer) to the center of the membrane of the DNA preparation tube, and let stand at room temperature for 1min (preheating the EB buffer at 50°C may ha...

Embodiment 3

[0102] Example 3. Plasmid pACgp67B-Her2-scfv-protamine constructed by enzyme digestion and identification of Her2 single-chain fragment antibody and protamine polypeptide

[0103] Perform enzyme digestion to identify the pACgp67B-Her2-scfv-protamine plasmid fragment:

[0104] ①. The restriction site of BamHI (G^GATCC) is at 4259; the restriction site of XhoI (C^TCGAG) is at 1902; therefore, the size of the digested band is 2357bp, and the position is accurate. Such as figure 1 As shown, pACgp67B-Her2-scfv-protamine was identified by digestion with BamHI and XhoI. M is a marker, 1, 2, 3, 4, 5, 6, and 7 are amplified and extracted plasmid DNA from different clones. After enzyme digestion, there is a corresponding band above 2027bp, which is consistent with the position of the expected band.

[0105] ②. The enzyme cutting sites of HindIII (A^AGCTT) are at: 2, 5328, 6256, and 7292, so the sizes of the four bands after enzyme cutting are: 5326bp, 3423bp, 1036bp, 928bp, and the po...

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PUM

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Abstract

The present invention provides a single chain fragment antibody-polypeptide fusion protein and applications thereof, the protein is a fusion protein which can be combined with Her2 receptors and can carry anti-cancer siRNA drug, and the fusion protein can serve as the vector of the anti-cancer siRNA drug. The single chain fragment antibody is Her2-ScFv, and the polypeptide is nucleoprotamine fragment polypeptide. The sequence of the single chain fragment antibody-polypeptide fusion protein is shown as SEQ ID NO.1. The Her2 single chain fragment antibody and the nucleoprotamine polypeptide fusion protein, which are constructed by the present invention, can be combined with Her2 receptors and can carry the anti-cancer siRNA drug. The fusion protein of the present invention can directionally introduce the anti-cancer siRNA drug into target cancer cells, and a non-viral vector tool is developed to speed up the application of the RNAi technology in clinic.

Description

technical field [0001] The invention belongs to the technical field of biology and medicine, and specifically relates to a single-chain fragment antibody-polypeptide fusion protein and its application. Background technique [0002] Her2 is a marker of breast cancer progression. Recent studies have found that about 25-30% of breast cancer patients' cancer tissue cells express an epidermal growth factor receptor 2 (Human Epidermal Growth Factor Receptor 2, Her2), and Her2 protein is a molecular marker of breast cancer progression. [0003] At present, there are mainly anti-Her2 monoclonal antibodies, tyrosine kinase inhibitors and traditional antisense gene methods to treat breast cancer by blocking Her2 receptors. Among them, anti-Her2 monoclonal antibodies, such as monoclonal antibody Herceptin, work by blocking receptor proteins, but because breast cancer cells Her2 receptors are overexpressed at the gene level, new Her2 proteins are still being synthesized and transported...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/63C12N5/10A61K47/42A61K31/7088A61P35/00
Inventor 宋尔卫姚燕丹朱鹏程
Owner GUANGZHOU ANJIE BIOMEDICAL TECH CO LTD
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