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Preparation method for adenovirus antigen and rapid detection kit used for detecting adenovirus antibody and prepared by utilizing adenovirus antigen

An adenovirus and antigen technology, applied in the field of clinical medical detection, can solve the problems of cumbersome procedures, high cost, limited output, etc., and achieve the effects of high immunogenicity, high specificity and sensitivity, and high expression efficiency

Inactive Publication Date: 2017-11-21
LANZHOU YAHUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The quality of adenovirus antibody detection reagents depends on the performance of the antigen. Due to the cumbersome procedures for extracting and preparing antigens from natural adenoviruses, the cost is high and the output is limited.

Method used

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  • Preparation method for adenovirus antigen and rapid detection kit used for detecting adenovirus antibody and prepared by utilizing adenovirus antigen
  • Preparation method for adenovirus antigen and rapid detection kit used for detecting adenovirus antibody and prepared by utilizing adenovirus antigen
  • Preparation method for adenovirus antigen and rapid detection kit used for detecting adenovirus antibody and prepared by utilizing adenovirus antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Recombinant expression, structure renaturation and purification of recombinant adenovirus penton protein antigen.

[0044] The adenovirus penton gene refers to the GenBank sequence KP270914.1, and the gene is selected for synthesis. The codon optimization of E. coli is carried out during the synthesis. The expression vector is pET30a, and the enzyme cutting site for connection is EcoR I / Xho I. The total target gene is 1635bp (544aa), transformed into BL21 (DE3), the expressed recombinant protein was 592aa in total, with a molecular weight of 67.6 kDa and an isoelectric point of 5.4. Recombinant proteins are expressed as inclusion bodies.

[0045] Construction and identification of recombinant expression vectors: EcoR I and Xho I were used to double-enzyme-digest the adenovirus penton target gene fragment and pET30a plasmid respectively, and after purification and recovery of the enzyme-digested products, they were ligated overnight at 16°C and then transformed...

Embodiment 2

[0048] Example 2 Preparation of a gold-labeled rapid detection kit for adenovirus antibody IgG / IgM.

[0049] The genetically recombinant adenovirus penton obtained above was used as a detection antigen, and the detection line was coated on a nitrocellulose membrane. figure 2 , prepare the adenovirus antibody gold label rapid detection reagent, its composition comprises: be provided with on the liner plate 10 the water-absorbing layer 4 of the sample loading end, the detection layer 8 and the water-absorbing layer 9, between the detection layer and the sample loading end water-absorbing layer 4 A gold-labeled anti-adenovirus antibody layer 5 is provided, and a detection line 7 and a quality control line 6 are coated on the detection layer 8 . Wherein, the water-absorbing layer 4 at the sample loading end and the water-absorbing layer 9 at the water-absorbing end are made of multi-layer filter paper: the detection layer 8 is a nitrocellulose membrane; the gold-labeled antibody ...

Embodiment 3

[0053] Example 3 Determination of Adenovirus Antibody.

[0054] Take 10 μl of serum or plasma sample and drop it into the sample well 2 of the detection plate 1, then add 100 μl of the sample diluent into the sample well 2, and observe the detection result in the observation window 3, and the observation result is valid within 20 minutes. If the sample contains anti-adenovirus antibody, two red lines appear in the detection line and quality control line in the observation window, and the test result is judged as positive; if the serum does not contain anti-adenovirus antibody, the quality control line in the observation window If a red line is seen at the position of the line, the test result is judged as negative; if no red line can be seen in the observation window, the test result is invalid.

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Abstract

The invention relates to an adenovirus artificial antigen prepared by gene engineering expression, and a method for preparing the adenovirus antigen, a method for detecting an adenovirus antibody rapidly, and a kit used for detecting the adenovirus antibody rapidly, wherein the adenovirus antigen has high specificity. The method for preparing the adenovirus antigen comprises the following steps: synthesizing an optimized adenovirus penton gene sequence artificially, constructing a prokaryotic expression vector, expressing the adenovirus penton antigen through escherichia coli, and performing renaturation on an inclusion body by adopting a dialysis method, a gradient dilution method and gel chromatography to obtain a recombined adenovirus penton antigen with a three-dimensional structure and immunocompetence. The method for detecting the adenovirus antibody rapidly comprises a step of applying the adenovirus penton antigen. The kit used for detecting the adenovirus antibody rapidly contains the adenovirus penton antigen, comprises a detection device which has a blood filtering membrane, and can be directly used for whole-blood detection.

Description

technical field [0001] The invention belongs to the field of clinical medical detection, relates to immunochromatographic detection technology, in particular to an adenovirus antigen used for diagnosing adenovirus infection, a method for preparing the antigen, and a rapid detection kit for detecting adenovirus antibody using the antigen. Background technique [0002] Adenovirus (Adenovirus, Adv) is one of the main pathogens of viral pneumonia around the world. Adenovirus infection in children or young adults only causes upper respiratory tract disease, but can cause severe acute adenovirus pneumonia in infants and young children. Adenoviral pneumonia was first reported by Chang et al. in 1958. Subsequent studies found that the pathogens causing adenoviral pneumonia in infants and young children are mainly adenovirus types 3 and 7. Mild and older children have a better prognosis. Severe infection and adenovirus The mortality rate of pneumonia with poor prognosis is high. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/075C07K1/16G01N33/569
CPCG01N33/56983
Inventor 李克生杜惠芬曾潮宁许菲菲范丽赟
Owner LANZHOU YAHUA BIOTECH
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