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EB virus BFRF3-BZLF1 fusion protein, gene, vector containing same, host cell, test strip and production method and application thereof

A technology of BFRF3-BZLF1 and Epstein-Barr virus, applied in the field of bioengineering, can solve the problems of not being able to obtain high sensitivity and high specificity at the same time, easily causing missed diagnosis, and increasing specificity, so as to achieve improved specificity and sensitivity, high specificity, Effects with low specificity

Active Publication Date: 2019-06-25
北京贝思泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

VCA is the latest antigen expressed after Epstein-Barr virus infection, and can exist in the host for life. Therefore, capsid antigen-immunoglobulin A (VCA-IgA) detection is often used for screening to exclude the diagnosis of NPC, and its sensitivity is higher than that of NPC. 90%, but its specificity is sometimes lower than 70%, which is likely to cause misdiagnosis during large-scale population screening; anti-EA antibodies are rare in healthy people, but their specificity increases in patients with nasopharyngeal carcinoma. Therefore, early Antigen-immunoglobulin A (EA-IgA) detection is currently used for the definitive detection of nasopharyngeal carcinoma. The specificity is higher than 90%, but the sensitivity is lower than 80%. It is easy to cause missed diagnosis in large-scale population screening
Therefore, Epstein-Barr virus-capsid antigen-immunoglobulin A (EBV-VCA-IgA), Epstein-Barr virus-early antigen-immunoglobulin A (EBV-EA-IgA) detection cannot simultaneously obtain high sensitivity and high specificity

Method used

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  • EB virus BFRF3-BZLF1 fusion protein, gene, vector containing same, host cell, test strip and production method and application thereof
  • EB virus BFRF3-BZLF1 fusion protein, gene, vector containing same, host cell, test strip and production method and application thereof
  • EB virus BFRF3-BZLF1 fusion protein, gene, vector containing same, host cell, test strip and production method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design and cloning of Epstein-Barr virus BFRF3-BZLF1 fusion protein

[0037] 1. Selection of amino acid sequence of fusion protein

[0038] The BFRF3 protein selects 83 amino acids at its N-terminal, the amino acid sequence is shown in SEQ ID NO: 3, and the sequence structure of the SEQ ID NO: 3 is as follows:

[0039] Met Ala Arg Arg Leu Pro Lys Pro Thr Leu Gln Gly Arg Leu Glu Ala AspPhe Pro Asp Ser Pro Leu Leu Pro Lys Phe Gln Glu Leu Asn Gln Asn Asn Asn Leu ProAsn Asp Val Phe Arg Glu Ala Gln Arg Ser Tyr Leu Val Phe Leu Thr Ser Gln PheCys Tyr Glu Glu Tyr Val Gln Arg Thr Phe Gly Val Pro Arg Arg Gln Arg Ala IleAsp Lys Arg Gln Arg Ala Ser Val Ala.

[0040] The BZLF1 protein selects 90 amino acids at its C-terminus, the amino acid sequence is shown in SEQ ID NO: 4, and the sequence structure of the SEQ ID NO: 4 is as follows:

[0041] Ala Arg Arg Thr Arg Lys Pro Gln Gln Pro Glu Ser Leu Glu Glu Cys AspSer Glu Leu Glu Ile Lys Arg Tyr Lys Asn Arg Val Ala Ser Arg L...

Embodiment 2

[0051] Example 2 Expression of Epstein-Barr virus BFRF3-BZLF1 fusion protein

[0052] 1. Construction of expression plasmid

[0053] The cloned plasmid was double-digested with NdeI and XhoI. The endonucleases NdeI and XhoI were both purchased from NEB Company. The excised target gene was connected to the pET-30a expression vector with ligase. The T4 DNA ligase enzyme provided by the company is the constructed expression plasmid.

[0054] Transform the competent cells E. coli BL21(DE3) with the expression plasmid for small-scale expression. The target protein band size is about 20KDa. Pick positive clones and divide them into plates on the bacterial culture dish, and perform small-scale expression again , pick positive clones to extract plasmids, carry out enzyme digestion identification, and send the identified positive samples to the sequencing company for sequencing to confirm the correctness of the expression plasmid construction, such as figure 1 It is a flowchart of th...

Embodiment 3

[0057] Example 3 Application of EB virus BFRF3-BZLF1 fusion protein in EB virus ELISA detection

[0058] ELISA detection principle: use the recombinant antigen prepared by the present invention to coat the plate, add the serum to be tested, after the antibody in the serum is combined with the plate antigen, use the enzyme-labeled anti-human secondary antibody to combine with the antibody, and develop the color measurement result.

[0059] (1) Antigen coating: the purified recombinant BFRF3-BZLF1 fusion protein was coated with diluent (35mM NaHCO 3 ,15mM Na 2 CO 3 , pH9.6) was diluted to a concentration of 100 μg / mL. Add 100 μL to each well on the ELISA plate, 37°C, 3h.

[0060] (2) Plate washing: wash the microtiter plate 5 times with PBST (PBS containing 0.05% Tween-20).

[0061] (3) Blocking: add 100 μL / well of 5% bovine serum albumin (BSA) blocking solution (prepared in PBST), and block at 37° C. for 1 hour.

[0062] (4) Plate washing: Same as above.

[0063] (5) Prim...

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Abstract

The invention provides an EB virus BFRF3-BZLF1 fusion protein, a gene, a vector containing the BZLF1 fusion protein, a host cell, a test strip, a production method and application thereof. The EB virus BFRF3-BZLF1 fusion protein is obtained by connecting the EB virus BFRF3 protein or an EB virus BFRF3 protein fragment with an EB virus BZLF1 protein or EB virus BZLF1 protein fragment through a connecting peptide, and preferably, the EB virus BFRF3-BZLF1 fusion protein has an amino acid sequence shown in SEQ ID NO:1. The EB virus BFRF3-BZLF1 fusion protein can be used as a target antigen for detecting an EB virus, provides a basis for screening and early diagnosing nasopharyngeal carcinoma, infectious mononucleosis and Burkitt lymphoma, and has the advantages of simplicity, rapidness, sensitivity and specificity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an Epstein-Barr virus BFRF3-BZLF1 fusion protein, a gene, a carrier containing the same, a host cell, a test strip and a production method and application thereof. Background technique [0002] Epstein-Barr (EB) virus is the only lymphofollicular virus in the Gamma subfamily of the Herpesviridae family that can cause human infection. Epstein-Barr virus has the characteristics of B lymphocytes, can establish a recessive infection in B lymphocytes, and stimulate cell proliferation and transformation. In recent years, it has been found that there are structures similar to Epstein-Barr virus receptors on the surface of epithelial cells in the parotid duct, oropharynx and external os of the cervix. Epstein-Barr virus is closely related to human infectious mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) and other diseas...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21G01N33/569G01N33/574G01N33/52C12R1/19
Inventor 陈斯勇
Owner 北京贝思泰生物科技有限公司
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