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HIV (Human Immunodeficiency Virus) recombinant antigen, expression gene, expression vector and HIV detection kit

A technology for recombinant antigen and gene expression, which can be used in recombinant DNA technology, antibody mimics/scaffolds, and introduction of foreign genetic material using vectors, which can solve the problem of high risk of missed detection of HIV-O subtypes.

Active Publication Date: 2017-05-10
FAPON BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the distribution of HIV-O subtypes in China is very small, with the continuous strengthening of the flow of people at home and abroad, the infection of HIV-O subtypes in China has a tendency to increase; on the other hand, domestic AIDS testing reagents will also be exported to HIV-O Countries and regions with severe subtype infection, so the risk of missed detection of HIV-O subtype by existing reagents is increasing

Method used

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  • HIV (Human Immunodeficiency Virus) recombinant antigen, expression gene, expression vector and HIV detection kit
  • HIV (Human Immunodeficiency Virus) recombinant antigen, expression gene, expression vector and HIV detection kit
  • HIV (Human Immunodeficiency Virus) recombinant antigen, expression gene, expression vector and HIV detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1, the preparation of coated antigen and labeled antigen

[0068] Using genetic engineering technology, a large number of molecular biology analysis software analyzes and screens the gp41 protein of HIV-1, the gp41 protein of O subtype, and the dominant epitope gene segment of gp36 protein of HIV-2. The sequence is SEQ ID No. .1 (named HI), SEQ ID No.2 (named HII), SEQ ID No.3 (named O), design primers to amplify the target fragment, and perform point mutation on the HI fragment in order to improve the specificity of the kit. The mutation sites are HIV-1gp41T67A and D121E, and the sequence after mutation is named as HIA (SEQ ID No.4). The upstream primer of the HIA fragment has a BamHI site, the downstream primer has an EcoRI restriction site, the upstream primer of the HⅡ fragment has an EcoRI site, the downstream primer has a SalI restriction site, and the O fragment upstream primer has a SalI site, The downstream primer has an XhoI restriction site, the P...

Embodiment 2

[0072] Example 2, establishment of anti-human HIV-1 P24 hybridoma cell line and preparation of monoclonal antibody thereof

[0073] 1. Immunization of mice with recombinant P24 antigen

[0074] Dilute the P24 recombinant antigen (P24-Ag, Feipeng Biological Co., Ltd.) to 1.0 mg / mL, mix it with Freund's complete adjuvant (Sigma-Aldrich, product number: F5881) in equal volumes, and fully emulsify to obtain an oily emulsion . The emulsion was subcutaneously administered to the dorsal site of BALB / c mice (Guangdong Provincial Medical Experimental Animal Center: No. 119, Poyang Road, Huangqi, Nanhai, Foshan City, Guangdong Province, 6-week-old female, 5 mice) at a dose of 0.2 mL. Fourteen days after the first immunization, the intraperitoneal booster immunization was performed, that is, the same amount of antigen was mixed with an equal volume of Freund’s incomplete adjuvant (Sigma-Aldrich, F5506). The titer was determined by the method, if the titer is higher than 1:10000, it can...

Embodiment 3

[0097] Embodiment 3, the preparation and debugging of HIV detection kit

[0098] 1. Preparation of HRP-labeled antigen

[0099] Using NaIO 4 oxidation method. Weigh 8mg of horseradish peroxidase (HRP, British Biozyme laboratories company, article number: HRP4) and dissolve it in 0.4mL ultrapure water, then slowly add 20mg / mL NaIO freshly prepared in 0.4mL ultrapure water 4Solution, stirred gently at room temperature for 40 minutes in the dark, then added 48 μL of ethylene glycol (dissolve 8 μL of ethylene glycol in 40 μL of distilled water) solution, and stirred at room temperature for 40 minutes in the dark. Then immediately add 1 mL of the purified labeled antigen GST-HIA-HII-O obtained in Example 1 at 2 mg / mL, which was dialyzed against 20 mM, pH 9.51 carbonate buffer solution for 2 hours, and protected against light at 4°C to 20 mM, pH 9 .51 carbonate buffer dialyzed overnight. The next day, add 80 μL of freshly prepared 5 mg / mL NaBH dropwise to the mixture 4 solution...

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Abstract

The invention discloses an HIV (Human Immunodeficiency Virus) recombinant antigen capable of detecting an HIV-O subtype, an expression gene and an expression vector which are used for expressing the HIV recombinant antigen, and an HIV detection kit containing the HIV recombinant antigen. The HIV recombinant antigen comprises an HIV-1 type segment, an HIV-2 type segment and an HIV-O subtype segment, which are connected in sequence; the HIV-O subtype segment is a polypeptide obtained by encoding a nucleotide sequence shown as SEQ ID No.3. The HIV recombinant antigen contains an HIV-O segment, so that the HIV recombinant antigen can be used for detecting the HIV-O subtype. In a specific test, the HIV detection kit containing the HIV recombinant antigen has relatively high specificity and sensitivity in the aspect of detecting the HIV-O subtype.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an HIV recombinant antigen, expressed gene, expression vector and HIV detection kit. Background technique [0002] Since Acquired Immunodeficiency Syndrome (AIDS) was named in 1982, HIV (Humanimmunodeficiency virus) infection has spread widely around the world, seriously threatening people's life and health. Therefore, HIV infection must be monitored. In order to detect infected persons as soon as possible, the "Notice of the State Council on Further Strengthening AIDS Prevention and Control Work" Guofa [2010] No. 48 clearly stated that the construction of monitoring and testing networks should be further strengthened and the scope of testing services should be expanded by relying on existing medical and health resources to equip necessary equipment and personnel Promote the use of fast and simple detection methods to improve detection accessibility. In view of this, the development...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63G01N33/577G01N33/569C12N5/20C12R1/91
CPCC07K14/005C07K2319/00C12N2740/16022G01N33/56988G01N33/577
Inventor 李瑞净孟媛于秀玲王益琼林育佳孙康成
Owner FAPON BIOTECH INC
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