EF-Tu protein monoclonal antibody MAb of Brucella Melitensis and preparation method and application thereof

A monoclonal antibody, brucellosis technology, applied in the field of biology, can solve the problem of no safe and effective human vaccine

Active Publication Date: 2016-01-06
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although several vaccines are available for animal immuni

Method used

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  • EF-Tu protein monoclonal antibody MAb of Brucella Melitensis and preparation method and application thereof
  • EF-Tu protein monoclonal antibody MAb of Brucella Melitensis and preparation method and application thereof
  • EF-Tu protein monoclonal antibody MAb of Brucella Melitensis and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0039] The construction of embodiment 1 prokaryotic expression vector PET30a-EF-Tu

[0040] The primers designed by the inventor at first are as follows:

[0041] Upstream primer 1: 5'-TTGGATCCATGGCAAAGAGTAAGTTTGAAC-3', the nucleotide sequence of which is shown in SEQ ID NO.1,

[0042] Downstream primer 2: 5'-TTCTCGAGACTCGATGATCGACGAGACGAT-3', the nucleotide sequence of which is shown in SEQ ID NO.2;

[0043] Using the BM28 genome (GenBank sequence number: CP002459.1) as a template, after PCR amplification with the following primers, the EF-Tu gene is obtained; the specific process is as follows:

[0044] The reaction conditions for PCR are:

[0045] The reaction system is 50 μl:

[0046] 10×PCRBuffer (Mg2+) 5 μl, dNTPMixture (2mM) 5 μl, upstream and downstream primers 1 μl (10 μmol / L), Blendtaq-plus (Promega) 0.5 μl, BM28 genomic DNA template 1 μl, sterilized deionized water to make up to 50 μl.

[0047] The PCR amplification program is:

[0048] Pre-denaturation at 94°C...

Embodiment 2

[0051] Example 2 Expression and Purification of Brucella Malta Type EF-Tu Protein Antigen

[0052] The positive clones obtained in Example 1 were transferred into Rosetta / BL21 (DE3), and the culture medium was kana-resistant LB plate.

[0053] Pick a single colony and culture it overnight in LB liquid, transfer it to fresh liquid LB medium at 1:50 (v / v), culture with shaking at 37°C until OD600 is approximately equal to 0.6-0.8, add IPTG to a final concentration of 1 mM, and continue to culture for 4 hours ;

[0054] The bacterial culture induced to express for 4h was centrifuged at 4000r / min for 20min, and the bacteria were collected, resuspended in PBS (pH7.4) at 3mL / g wet weight of the bacteria, and 80μL of 10mg / mL lysozyme (Soleibo) and 8μL of 50mmol / L Phenylmethylsulfonyl fluoride (PMSF), act at 37°C for 0.5h, add deoxycholic acid 20mg / g bacterial cell wet weight, room temperature until the bacterial liquid is viscous, add 1mg deoxyribonuclease I (Biyuntian Biotechnology...

Embodiment 3

[0057] Embodiment 3 animal immunization

[0058] The purified protein was fully mixed and emulsified with an equal volume of Freund's complete adjuvant, and 6-week-old Balb / c mice (100 μg / mouse) were immunized intraperitoneally. Two weeks later, the protein was emulsified with Freund's incomplete adjuvant. Two weeks after three immunizations, one week after the third immunization, the whole blood of BALB / c mice was collected from the tail vein, the serum was naturally separated, and the antibody titer in the serum was determined by indirect ELISA (96 wells coated with recombinant protein antigen ELISA reaction plate), if the serum titer is higher than 1:10000, then the mouse splenocytes can be taken for the next cell fusion test. Booster immunization: In order to increase the titer of the antibody, the recombinant protein antigen was dissolved in PBS buffer 3 days before the fusion, and 50ug of the tail vein was taken to immunize BALB / c mice again.

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Abstract

The invention provides an EF-Tu protein monoclonal antibody MAb of Brucella Melitensis and a preparation method and application thereof. An EF-Tu gene is cloned to a pET30a vector to construct an EF-Tu protein for expressing an His-carrying label in an prokaryotic expression vector, the purified EF-Tu protein is used for immunizing a Balb/c mouse, the mouse spleen cell and myeloma SP2/0 cell are fused to prepare a hybridoma cell, and the anti-Brucella Melitensis EF-Tu MAb is obtained by specific screening, in-vitro culturing and purifying. An ELISA plate is coated by adopting EF-Tu purified soluble protein for prokaryotic expression of a GST-carrying label during screening, so that the condition that MAb recognizing His label protein is screened can be avoided, the soluble GST-EF-Tu protein has activity close to the natural Bacterium burgeri elongation factor Tu protein, and the specificity of the hybridoma cell is ensured. The Western blot result shows that the MAb can react with the GST or His label-carrying Bacterium burgeri protein antigen rather than the GST label or Escherichia coli thalli protein, and the antibody MAb has strong specificity.

Description

technical field [0001] The invention relates to the field of biology, and specifically provides an EF-Tu protein monoclonal antibody MAb of Brucella malta, a preparation method and application thereof. Background technique [0002] Brucellosis, also known as "Mediterranean relaxation fever" or "Maltese fever", referred to as "brucellosis", is an important zoonotic infectious disease caused by Brucella infection. Since the 1990s, with the development of animal husbandry, the infection rate of brucellosis among people and herds has increased, and the number of cases has increased year by year. About 500,000 people in the world are infected every year. The loss is immeasurable, which seriously threatens human health and the development of animal husbandry. At present, although several vaccines have been used for animal immunization, there is no safe and effective vaccine for human use. In order to develop a safer and more effective brucellosis vaccine, it is urgent to strengt...

Claims

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Application Information

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IPC IPC(8): C07K16/12A61K39/395A61P31/04G01N33/577
Inventor 王方昆赵孝民李宏梅刘思当赵宁宁
Owner SHANDONG AGRICULTURAL UNIVERSITY
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