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A recombinant protein containing human IgG1fc and the C-terminus of mannan-binding lectin

A technology of mannan and recombinant protein, which is applied in the field of recombinant protein, can solve the problems of low expression of active protein and delay in clinical diagnosis, and achieve good application prospects and the ability to enrich pathogens

Active Publication Date: 2021-05-04
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Based on the increase of blood pathogen infection in hospitalized patients, the delay of clinical diagnosis, the defects of existing detection methods, the low expression level of active protein, and the uncertainty of the existing expression system, the purpose of the present invention is to obtain the recombinant protein by nuclear The nucleotide sequence is optimized to significantly increase its expression in mammalian cells and have better activity in enriching pathogens

Method used

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  • A recombinant protein containing human IgG1fc and the C-terminus of mannan-binding lectin
  • A recombinant protein containing human IgG1fc and the C-terminus of mannan-binding lectin
  • A recombinant protein containing human IgG1fc and the C-terminus of mannan-binding lectin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Codon optimization and synthesis of human MBL-IgG1Fc recombinant protein gene.

[0040] First, design and express the human MBL-IgG1Fc recombinant protein gene sequence structure, such as figure 1As shown, the human interleukin-2 (IL-2) signal peptide sequence was added at the N-terminal, and the enzyme cleavage sites at both ends were NheI and NotI. Then, use the JCat (JAVA Codon Adaptation Tool) tool (http: / / www.jcat.de / Result.jsp) to optimize the codons of the gene of the MBL-IgG1Fc recombinant protein, and then perform artificial Adjusted to make it more suitable for expression in 293F cells. See SEQ ID NO:1 for the codon-optimized recombinant protein MBL-IgG1FcYU gene sequence, see SEQ ID NO:2 for the amino acid sequence, and see SEQ ID NO:3 for the unoptimized recombinant protein MBL-IgG1Fc gene sequence. After codon optimization, the overall nucleotide sequence of the recombinant protein changed by 16.5%, while the amino acid sequence remained unchan...

Embodiment 2

[0055] Example 2. Detection of the binding activity of recombinant protein activity and pathogenic bacteria.

[0056] It can be seen from the results of Example 1 that the concentration of purified protein expressed in cells transfected with pCDNA3.1-MBL2-IgG1FcYU plasmid is significantly higher than that expressed in cells transfected with pCDNA3.1-MBL2-IgG1FcYU plasmid. In order to further prove that the protein expressed by the pCDNA3.1-MBL2-IgG1FcYU plasmid-transfected cells has good pathogen-binding activity, further activity identification of the protein expressed by the pCDNA3.1-MBL2-IgG1FcYU plasmid-transfected cells will be carried out below.

[0057] 1. WB detection of the combination of recombinant protein and Candida.

[0058] Draw 6×10 7 CFU Candida, washed 3 times with PBS, 3000rpm, 1 minute, centrifuged at room temperature, resuspended in 500μl PBS, added 2ul 1M CaCl and 10μl 400μg / ml MBL recombinant protein, incubated at room temperature for 30 minutes. Centrif...

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Abstract

The invention provides a recombinant protein containing human IgG1 Fc and mannan-binding lectin C-terminus and a polynucleotide whose coding sequence is optimized. The invention also provides a preparation method for expressing the recombinant protein. The purified recombinant protein expressed by the plasmid-transfected cells containing the sequence-optimized polynucleotide has a concentration of 1.50 mg / ml, which is 3 times that of the protein expressed by the codon-unoptimized coding sequence. The recombinant protein has good binding activity to the 5 Candida standard strains and clinical strains that account for more than 90% of blood fungal infections, which proves that the recombinant protein can bind to the 5 Candida strains, and the combination is due to recombination Mediated by the CRD region of the protein MBL. The good Candida binding ability shows that the recombinant protein has a good ability to enrich pathogens, and has a good application prospect in microbial detection.

Description

technical field [0001] The invention discloses a recombinant protein and belongs to the technical field of polypeptides. Background technique [0002] At present, there are more and more blood pathogenic bacterial infections in hospitalized patients, and the gold standard for diagnosis of blood pathogenic bacterial infections is still blood culture, which takes a long time and often delays diagnosis and treatment. Therefore, the development of faster diagnostic techniques is an urgent problem to be solved. . Molecular detection methods that do not depend on culture, especially nucleic acid detection, have become a hot research topic at present. However, the content of pathogens in blood is low. If the whole blood genome is directly extracted for nucleic acid detection, the positive rate is extremely low. This requires preliminary enrichment of pathogens in blood to greatly increase the detection rate of pathogenic nucleic acids. There are many methods for enriching pathoge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10
CPCC07K14/7056C07K2319/30C12N15/85C12N2800/107C12N2800/22
Inventor 陈小萍陈晓丽李文革卢金星
Owner 北京卓诚惠生生物科技股份有限公司