PATENT CLAIM ANALYSIS

Application Number: 15763585
Application Type: Utility
Filing Date: 2018-03
Publication Date: 2018-11
Patent Classification: ["435", "440000"]

Abstract:
The present invention provides a construction method of cell models for detecting pyrogens, cell models and pyrogen detection kits. By inducing double-bond breakage in the specific positions of genomes using CRISPR/CAS9, performing directed knock-in of TLR4 and CD14-MD2 on two chromosomes in a cell line based on the principle of homologous recombination and repair, and tracing using green fluorescent protein GFP and red fluorescent protein RFP respectively, we successfully construct the TLR4/CD14/MD2 directed knock-in and fluorescence-traceable cell model; upon the exposure to LPS, the release of IL-6 and TNF-a cytokines in the cell model can be detected by ELISA, Western Blot, mass spectrometry and magnetic bead method; and the cell model has good stability and high sensitivity, and the lowest detection limit can reach 0.005 EU/mL, which is far lower than that of the Limulus amebocyte lysate endotoxin test method (0.025 EU/mL).

Claim (Index 1):
A construction method of a cell model for detecting pyrogens, including the following steps of:\n 1) assembling TLR4 genome into an expression vector capable of expressing a green fluorescent protein, to construct a recombinant plasmid containing TLR4 gene; 2) knocking the TLR4 gene in a directed manner into an introne of a gene on a chromosome of a cell line by the CRISPR/CAS9 method, to obtain a cell line capable of expressing the TLR4 gene stably; 3) segregating CD14 and MD2 genes from red fluorescent protein RFP gene using two different 2A peptides, to construct a recombinant plasmid containing CD14 and MD2 genes and capable of expressing a red fluorescent protein; and 4) knocking the CD14 and MD2 genes in another introne of a gene on another chromosome than the chromosome on which the TLR4 gene is knocked-in in step 2) by the CRISPR/CAS9 method, to obtain a cell line capable of expressing the TLR4, CD14 and MD2 genes stably and capable of expressing green and red fluorescent proteins, namely the cell model.

Metadata:
- Claim Count in Document: 27.0
- Percentile: 90.0
- Lexical Diversity: 1.53922
- Patent Class: 435.0
- Transitional Phrase Type: none
- Component Type: 0
- Foreign Priority: True
- Related Applications: ['10432236', '15148663', '14343974', '11613866', '13046562']

Analysis Scores:
- 35 USC 101 Eligibility (BERT): 0.6269934424982032
- 35 USC 102 Novelty (BERT): 0.4945707400512759
- Combined Prediction Score: 0.6137511722535105
- Mean Citation Score: 247.929142
- Max Citation Score: 255.49191
- Similarity Product: 199.5954252067262

Labels:
- Claim Label 101: 1
- Claim Label 102: 1
- Claim Label 103: 0
- Claim Label 112: 1
- Combined Label: 1
- Label 101 Adjusted: 1

Dataset: test