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Quantitative detection method of hpv l1 protein

A protein and known quantity technology, applied in the field of virology, can solve the problems that affect the accuracy, cannot directly measure the samples adsorbed with adjuvants, limit the throughput, etc., and achieve high-precision results

Active Publication Date: 2021-06-15
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, the method uses type-specific antibodies to detect each type of antigen, which greatly limits the throughput of the method
In addition, since there is no unified standard antibody at present, manufacturers use their own antibodies to detect their antigens, making it difficult to unify the quantitative results, which greatly limits the quality control and supervision of HPV preventive vaccines
In addition, the ELISA method cannot directly measure the sample adsorbed with the adjuvant, and the adjuvant needs to be desorbed before the measurement. However, the desorption rate of the desorption step greatly affects the accuracy of the method.

Method used

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  • Quantitative detection method of hpv l1 protein
  • Quantitative detection method of hpv l1 protein
  • Quantitative detection method of hpv l1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Example 1. Identification of HPV16 L1 and HPV18 L1 characteristic peptides

[0147] 1.1 Determination of candidate characteristic peptides

[0148] The inventors first selected trypsin that specifically cleaves the C-terminus of lysine and arginine, and used the PeptideMass module of ExPASy software to simulate the enzymes of HPV16 L1 and HPV18 L1 protein sequences (provided by Jiangsu Ruike Biotechnology Co., Ltd.) Then use the ClustalW module of Bioedit software to combine the above-mentioned simulated enzymolysis fragments with the L1 proteins involved in the types of HPV preventive vaccines (including HPV6, HPV11, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59) were compared to obtain peptides (ie, candidate type-specific signal peptides / characteristic peptides) that only exist in theoretical hydrolysis products of HPV16 or HPV18 L1 proteins. The results are shown in Table 2 and Table 3 respectively. In theory, HPV16 L1 has 21 candidate type-s...

Embodiment 2

[0160] Example 2. Mass Spectrometry Quantitative Detection Method for Single Type HPV L1

[0161] In this example, the content of the HPV16 characteristic peptide and HPV18 characteristic peptide obtained in Example 1 was measured by isotope dilution method, so as to realize the quantification of HPV16 L1 protein and HPV18 L1 protein.

[0162] 2.1 Characteristic peptides and internal standard peptides

[0163] The sequences of the characteristic peptides of HPV16 L1 protein and HPV18 L1 protein and the corresponding isotope-labeled peptides (internal standard peptides) are shown in Table 4. The above polypeptides were all synthesized by China GL Biochem LTD and packaged into 1 mg / bottle.

[0164] Table 4: Type-specific peptides and internal standard peptides of HPV16 L1 and HPV18 L1

[0165]

[0166] 2.2 Standard curve

[0167] A mixed solution of HPV16 characteristic peptide and HPV18 characteristic peptide was prepared as an external standard, the concentration of both...

Embodiment 3

[0176] Example 3. Determination of HPV16 L1 and HPV18 L1 protein content in commercial products

[0177] The inventors used the isotope dilution mass spectrometry (abbreviated as IDMS) described in Example 2 to analyze four parts of HPV16 L1 stock solution before monovalent adsorption (Y1-16, Y2-16, E1-16 and I1-16), four parts of HPV18 L1 Stock solution before monovalent adsorption (Y1-18, Y2-18, E1-18 and I1-18), a part of HPV16 L1 stock solution after monovalent adsorption (E1-16-Al), and commercial vaccine Cervarix (purchased from GlaxoSmithKline (GSK)), Gardasil (Gardasil) and Gardasil 9 (Gardasil 9) (purchased from Merck & Co) in the HPV16 L1 and HPV18 L1 protein content were detected; and, while using The conventional BCA method and Bradford method in the field were used to measure the protein content of the above samples. The detection results of HPV16 L1 protein content are as follows: image 3 , Table 6, the detection results of HPV18 L1 protein content are as foll...

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Abstract

The present invention relates to the field of virology. In particular, the present invention relates to a characteristic peptide for quantitative detection of HPV16 L1 protein and HPV18 L1 protein, and a method for quantitative detection of HPV16 L1 protein and HPV18 L1 protein.

Description

technical field [0001] The present invention relates to the field of virology. In particular, the present invention relates to a characteristic peptide for quantitatively detecting HPV16 L1 protein and HPV18L1 protein, and a method for quantitatively detecting HPV16L1 protein and HPV18 L1 protein. Background technique [0002] Human papillomavirus (HPV) belongs to the Papillomaviridae Papillomavirus genus, is a class of non-enveloped epitheliophilic small double-stranded DNA virus. Natural HPV exhibits an iso-icosahedral symmetric structure with T=7. The HPV genome is about 7.2-8kb, including 8 open reading frames, encoding 6 early proteins and 2 late proteins. The late gene region (L region) is about 3000bp long and encodes two viral capsid proteins, namely, the major viral capsid protein L1 and the minor capsid protein L2. The L1 and L2 proteins together form the viral capsid at a ratio of 5:1 , is closely related to virus packaging, cell entry, and infection. L1 capsi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/025G01N27/62G01N33/68C12P21/06
CPCC07K14/005C12N2710/20022G01N27/62G01N33/6848
Inventor 王佑春宁婷婷孙姗姗李梦怡聂建辉黄维金曹进
Owner NAT INST FOR FOOD & DRUG CONTROL
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