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

A fusion protein and fragment technology, which is applied in the fields of biology and medicine, can solve the problems of weak gene expression effect and cannot meet clinical application, and achieve the effect of improving expression

Active Publication Date: 2011-08-03
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 bidirectional function of binding Her2 receptor and carrying siRNA drugs.

[0039] After finding the full-length sequence of the Her2 single-chain fragment antibody from the gene library, primer software was used to design the relevant primers.

[0040] Designed Her2-ScFv primers:

[0041] Positive primer: NcoI is an endonuclease

[0042] 5’-GGGCCATGGCCCAGGTGCAGCTGTTGCAGTCTGGGGCAGAG-3’

[0043] Reverse primer: NotI is an endonuclease

[0044] 5’-TTGCGGCCGCTCCGGAATTCACCTAGGACGGTCAGCTTGGTCCC-3’

[0045] Designed protamine peptide primers:

[0046] Positive primer: NotI is an endonuclease

[0047] 5’-CCGGAGCGGCCGCAATGGCCAGGTACAGATGCTG-3’

[0048] Reverse primer: PpuM I as an endonuclease, stop codon and 6 histidines

[0049] 5’-GCCGGGTCCCAGGAAAGGATCAGATCTGCATTAATGGTGGTGGTGATGATGAGATCTGTGTCTTCTACATCTCGGTCTG-3’

[0050] The relevant target fragments are cloned by PCR. The PCR reaction system of Her2-ScFv is 1:50ul system:

[0051] ddH 2 O 37ul

[005...

Embodiment 2

[0101] Example 2 Small amount extraction of pACgp67B-Her2-scfv-protamine plasmid

[0102] The bacteria were collected by centrifugation at 12000g×1min. (Generally, 3-4ml bacterial solution is used for one part extraction), and the supernatant is discarded. Add 250ul ice-bath Buffer S1 (containing RnaseA), vortex and shake 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×10min. Take the supernatant, pass through the DNA preparation tube, and discard the filtrate at 12000g×1min. Add 700ul BufferW1, 12000g×1min to the DNA preparation tube, and discard the filtrate. Add 500ul Buffer W2, 12000g×1min to the DNA preparation tube, and discard the filtrate. Centrifuge again at 12000g×1min. Add 40ul ddH2O (or EB buffer) to the center of the membrane of the DNA preparation tube, and let it stand at room temperature for 1 min (preheating EB buffer at 50°C...

Embodiment 3

[0103] Example 3. Restriction restriction identification of Her2 single-chain fragment antibody and the plasmid pACgp67B-Her2-scfv-protamine constructed by the protamine polypeptide

[0104] Restriction digestion to identify pACgp67B-Her2-scfv-protamine plasmid fragment:

[0105] ①. The restriction site of BamHI (G^GATCC) is at: 4259; the restriction site of XhoI (C^TCGAG) is at: 1902; so the size of the band after restriction is: 2357bp, 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, 7 are different clones of bacteria amplified and extracted plasmid DNA. After restriction digestion, there is a corresponding band at 2027 bp above, which is consistent with the expected band position.

[0106] ②. The restriction site of HindIII (A^AGCTT) is at: 2, 5328, 6256, 7292, so the size of the four bands after restriction is: 5326bp, 3423bp, 1036bp, 928bp, the position is accurat...

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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 directionallyintroduce 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 sign of worsening breast cancer. Recent studies have found that about 25-30% of breast cancer patients’ cancer tissue cells express a Human Epidermal Growth Factor Receptor 2 (Her2), and Her2 protein is a molecular marker of breast cancer deterioration. [0003] At present, the main ways to treat breast cancer by blocking Her2 receptors are anti-Her2 monoclonal antibodies, tyrosine kinase inhibitors and traditional antisense gene methods. Among them, anti-Her2 monoclonal antibodies, such as monoclonal antibody Herceptin, work by blocking the receptor protein, but because the Her2 receptor in breast cancer cells is overexpressed at the gene level, new Her2 proteins are still being synthesized and delivered continuously. To the cell membrane to...

Claims

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Application Information

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Patent Type & Authority Patents(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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