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Method for efficiently and quantitatively detecting PD-1 level in extracellular vesicles, ELISA kit and use method

A PD-1, quantitative detection technology, applied in the field of molecular biology and biology, can solve the problems of unfavorable clinical application, lack of standardized operation procedures, unclear and other problems

Pending Publication Date: 2020-08-14
珈泌生物科技(武汉)有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ultracentrifugation is time-consuming and laborious, lacks standardized operating procedures, and requires large-scale ultracentrifugation equipment, which is not conducive to clinical application; more importantly, studies have confirmed that the efficiency of ultracentrifugation for separating extracellular vesicles is less than 30%. , whether the level of PD-1 in these 30% EVs can accurately reflect the level of PD-1 in total EVs is still unclear

Method used

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  • Method for efficiently and quantitatively detecting PD-1 level in extracellular vesicles, ELISA kit and use method
  • Method for efficiently and quantitatively detecting PD-1 level in extracellular vesicles, ELISA kit and use method
  • Method for efficiently and quantitatively detecting PD-1 level in extracellular vesicles, ELISA kit and use method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Construction of CD63-PD-1 fusion protein

[0045] 1. According to PubmedGene query to human CD63 gene sequence (Gene ID: 967), select the Ala103 to Val203 translation fragment; PD-1 gene sequence (Gene ID: 5133), select the Leu125 to Gln167 fragment. Design the His protein tag sequence, start codon, and stop codon; insert the connexin sequence between the two sequences

[0046] (GGTGGTGGTGGTAGCGGTGGTGGCGGTAGTGGTGGTGGTGGTAGC), introduce EcoR1, Xhol double restriction sites, design a pair of positive and negative primers (Table 1), and perform PCR amplification to obtain the CD63-PD-1 gene fragment. .

[0047] Table 1. CD63-PD-1PCR primers

[0048]

[0049] 2. Ligate the CD63-PD-1 gene fragment with the Pet28a plasmid that has been digested by Ecorl and Xhol through Exnase recombinase (Novazyme, Cat. No. C113-01), and obtain the recombinant plasmid CD63-PD after transformation and screening -1, its nucleic acid sequence is shown in SEQ ID No: 1, and its a...

Embodiment 2

[0056] Example 2: Construction of CD9-PD-1 fusion protein

[0057] 1. According to Pubmed Gene query to the human CD9 gene sequence (Gene ID: 928), select the Ser112 to Ile195 translation fragment; PD-1 gene sequence (Gene ID: 5133), select the Leu125 to Gln167 fragment. Design the His protein tag sequence, start codon, and stop codon; insert the connexin sequence between the two sequences

[0058] (GGTGGTGGTGGTAGCGGTGGTGGCGGTAGTGGTGGTGGTGGTAGC), introduce EcoR1, Xhol double restriction sites, design a pair of positive and negative primers (Table 2), and perform PCR amplification to obtain the CD3-PD-1 gene fragment.

[0059] Table 2 CD9-PD-1PCR primers

[0060]

[0061] 2. Ligate the CD9-PD-1 gene fragment with the Pet28a plasmid that has been digested by Ecorl and Xhol through Exnase recombinase (Novazyme, Cat. No. C113-01), and obtain the recombinant plasmid CD9-PD after transformation and screening -1, its nucleic acid sequence is shown in SEQ ID No: 2, and its amino ...

Embodiment 3

[0068] Example 3: Construction of CD81-PD-1 fusion protein

[0069] 1. According to the PubmedGene query of the human CD81 gene sequence (Gene ID: 975), select the translated fragment from Phe113 to Lys201; PD-1 gene sequence (Gene ID: 5133), select the fragment from Leu125 to Gln167. Design the His protein tag sequence, start codon, and stop codon; insert the connexin sequence between the two sequences

[0070] (GGTGGTGGTGGTAGCGGTGGTGGCGGTAGTGGTGGTGGTGGTAGC), introduce EcoR1, Xhol double restriction sites, design a pair of forward and reverse primers (Table 3), and perform PCR amplification to obtain the CD3-PD-1 gene fragment.

[0071] Table 3. CD81-PD-1PCR primers

[0072]

[0073] 2. Ligate the CD81-PD-1 gene fragment with the Pet28a plasmid that has been digested by Ecorl and Xhol through Exnase recombinase (Novazyme, Cat. No. C113-01), and obtain the recombinant plasmid CD81-PD after transformation and screening -1, its nucleic acid sequence is shown in SEQ ID No: 3...

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Abstract

The invention relates to the field of molecular biology and biotechnology, and in particular relates to a method for efficiently and quantitatively detecting the PD-1 level in extracellular vesicles,an ELISA kit and a use method. The method comprises the steps of capturing extracellular vesicles of human body fluid by utilizing the specificity of an anti-CD63 antibody, an anti-CD9 antibody or ananti-CD81 antibody; using the anti-PD-1 antibody for detecting the PD-1 protein level in the extracellular vesicles; and using the constructed corresponding CD63-PD-1, CD9-PD-1 or CD81-PD-1 fusion protein as a standard substance to realize the quantitative detection of the PD-1 in the extracellular vesicles. According to the invention, quantitative detection of the extracellular vesicles of a complex structure is realized; and meanwhile, the fusion proteins CD63-PD-1, CD9-PD-1 and CD81-PD-1, which are independently constructed, are used as standard proteins, so that the detection result is characterized by the specific protein concentration.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a method for efficiently quantitatively detecting the level of PD-1 in extracellular vesicles, an ELISA kit and a use method. Background technique [0002] With the rising morbidity and mortality, malignant tumor is a major public health problem and has become the leading cause of death in our country. Although the level of surgery and the technology of radiotherapy and chemotherapy have made great progress in the past two decades, the recurrence rate and mortality of malignant tumors have not been significantly alleviated. Tumor immunotherapy, as an emerging treatment method, reactivates the human immune system to enable patients to rely on their own immunity to kill tumor cells and tumor tissues. Since anti-PD-1 inhibitors were first approved for marketing in 2014, after years of clinical trials, immunotherapy targeting the immune checkpoint PD-L1 / PD-1 signa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/574G01N33/543
CPCG01N33/54306G01N33/57484G01N33/581G01N2333/70521G01N2333/70596
Inventor 陈刚余自力刘金元林浩吴敏赵怡芳
Owner 珈泌生物科技(武汉)有限责任公司
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