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Preparation method of EB (Epstein-Barr) virus antigen and quick detection kit for detecting EB virus antibody prepared from antigen

A technology of Epstein-Barr virus and antigen, which is applied in the field of clinical medical detection, can solve the problems of unsuitable use, time-consuming, high detection conditions and technical requirements, etc., and achieve the effect of obvious effect, high antigen yield and high immunogenicity

Inactive Publication Date: 2016-12-07
LANZHOU YAHUA BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is time-consuming and requires special tissue culture conditions, so it is not suitable for clinical detection; the commonly used methods for detecting viral nucleic acids include nucleic acid hybridization and RT-PCR methods, which detect viral gene nucleic acids and viral genome transcripts in diseased tissues. The detection conditions and technical requirements are relatively high, so it is not suitable for routine clinical detection; serological diagnosis mainly detects the presence of specific antibodies such as CA-IgM / IgG antibodies and anti-nuclear antigen antibodies in serum by enzyme-linked immunosorbent assay and other methods , due to its advantages of high sensitivity, good specificity and easy operation, it is one of the most commonly used methods in laboratories at present, and has certain reference value for the diagnosis of diseases

Method used

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  • Preparation method of EB (Epstein-Barr) virus antigen and quick detection kit for detecting EB virus antibody prepared from antigen
  • Preparation method of EB (Epstein-Barr) virus antigen and quick detection kit for detecting EB virus antibody prepared from antigen
  • Preparation method of EB (Epstein-Barr) virus antigen and quick detection kit for detecting EB virus antibody prepared from antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Recombinant expression, structural renaturation and purification of recombinant Epstein-Barr virus self-fused capsid protein antigen

[0049] The Epstein-Barr virus capsid antigen P23-P18 fusion gene refers to the GenBank sequence V01555.2, selects the C-terminal gene sequences of the P23-encoded gene BLRF2 (amino acid 1-162) and the P18-encoded gene BFRF3, and the two genes are connected by a polypeptide linker (Gly 4 Ser) 3 The fusion gene was obtained by DNA sequence connection, and the whole gene was artificially synthesized. The expression vector was pET30a, and the enzyme cutting site was EcoR I / / XhoI. The total protein is 297aa, the molecular weight is 30.8 kDa, and the isoelectric point is 11.03. The recombinant protein is expressed in the form of inclusion body and can be purified by Ni column.

[0050] Construction and identification of recombinant expression vectors: EcoRI and XhoI were used to double-enzyme-digest the target gene fragment of EB ...

Embodiment 2

[0053] Example 2 Preparation of a gold-labeled rapid detection kit for Epstein-Barr virus antibody IgG / IgM

[0054] The self-fusion capsid protein p18-p23 of the genetically recombinant Epstein-Barr virus obtained above is used as a detection antigen, and the detection line is coated on a nitrocellulose membrane; refer to figure 2 , prepare EB virus antibody gold label rapid detection reagent, its composition comprises: on liner plate 10, be provided with sample loading end water-absorbing layer 4, detection layer 8 and water-absorbing layer 9, between detection layer and sample loading end water-absorbing layer 4 A gold-labeled anti-EB virus 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 gol...

Embodiment 3

[0058] The determination of embodiment 3 Epstein-Barr virus antibody

[0059] 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-EB virus antibody, two red lines will appear in the detection line and the quality control line in the observation window, and the test result is judged as positive; if the serum does not contain anti-EB virus 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 EB virus gene engineering artificial expression antigen and a method for preparing the antigen. The method comprises the following steps: artificially synthesizing a fused EB virus capsid proteantigen gene sequence, establishing a prokaryotic expression vector, expressing the EB virus capsid proteantigen in Escherichia coli, and renaturating the inclusion body by a dialyssis process, a gradient dilution process and gelchromatography to obtain the recombinant EB virus capsid proteantigen with the three-dimensional structure and immunocompetence. The invention also relates to a quick detection method for detecting an EB virus antibody. The method comprises the following step: using the EB virus capsid proteantigen. The invention also relates to a quick detection kit for EB virus antibody detection. The kit comprises the EB virus capsid proteantigen which can be directly used for whole blood detection. The kit comprises a rheumatism factor treatment pad which can be used for removing rheumatism factors in a sample and directly detecting IgM in the sample. The invention provides an EB virus antigen which has high specificity. The invention also provides a method for preparing the antigen, a method for quickly detecting the EB virus antibody and a kit for quickly detecting the EB virus antibody.

Description

technical field [0001] The invention belongs to the field of clinical medical detection, relates to immunochromatographic detection technology, in particular to an Epstein-Barr virus antigen for diagnosing Epstein-Barr virus infection, a method for preparing the antigen, and a rapid detection kit for detecting Epstein-Barr virus with the antigen. Background technique [0002] Epstein-Barr Virus (EBV) is human herpes virus type 4, which was first discovered by Epstein and Barr in 1964 when they studied African children's malignant lymphoma, and has the characteristics of human B lymphocytes. EBV is a DNA virus with a spherical shape and a diameter of 180-200nm, which replicates in B lymphocytes. Human is the host of EBV infection, and the virus is mainly transmitted through saliva. Asymptomatic infection mostly occurs in young children, and more than 90% of children aged 3 to 5 have been infected with EBV. Cellular immunity plays a key role in EBV infection, and the decline...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/03G01N33/577G01N33/569G01N33/558G01N33/543
CPCC07K14/005C12N2710/16021G01N33/543G01N33/558G01N33/56994G01N33/577G01N2333/03G01N2469/00
Inventor 李克生杜惠芬曾潮宁范丽赟许菲菲
Owner LANZHOU YAHUA BIOTECH
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