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Staphylococcus aureus-specific egg yolk immunoglobulin variable region single-chain antibody and application

An immunoglobulin and staphylococcus technology, applied in the preparation field of the antibody, can solve the problems of obtaining specific IgY active peptide, complicated separation and purification process, etc., achieves strong specificity, improves the level of early diagnosis and clinical treatment, and is good for The effect of the application foreground

Active Publication Date: 2015-10-07
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although IgY has many advantages, the specific IgY obtained by immunization of laying hens only accounts for 2-10% of the total IgY content, and the separation and purification process is cumbersome. Even if high-purity specific IgY is obtained after purification, it is difficult to digest in vitro and other means to obtain specific IgY active peptides

Method used

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  • Staphylococcus aureus-specific egg yolk immunoglobulin variable region single-chain antibody and application
  • Staphylococcus aureus-specific egg yolk immunoglobulin variable region single-chain antibody and application
  • Staphylococcus aureus-specific egg yolk immunoglobulin variable region single-chain antibody and application

Examples

Experimental program
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Effect test

Embodiment 1

[0022] 1. Antigen preparation, layer immunization and serum titer detection

[0023]Staphylococcus aureus (Staphylococcus aureus) CVCC 545 strain was purchased from China Center for Veterinary Microbiological Culture Collection. Transfer an appropriate amount of Staphylococcus aureus strains to 200ml TSB medium, culture overnight at 37°C, centrifuge at 4°C and 5000rpm for 10min, collect the bacteria, resuspend and wash with 0.01mol / l PBS three times, and then Inactivate with 0.5% formaldehyde at 37°C for 24 hours, take part of the bacterial solution and adjust to 10 with PBS 9 cfu / ml for future use, and the rest were freeze-dried to obtain freeze-dried bacterial cell powder.

[0024] Mix the above inactivated Staphylococcus aureus liquid with Freund's complete adjuvant (primary immunization) or Freund's incomplete adjuvant (second immunization and third immunization) in equal volumes, and emulsify by double push method to make a vaccine. 120-day-old healthy laying hens were ...

Embodiment 2

[0046] Example 2: Phage antibody blocking ELISA detection

[0047] A. Amplification of ScFv single phage:

[0048] From the plate of the last round of phage titer detection above, a single phage spot was randomly picked and placed in 100 μl of Escherichia coli BLT5403 (A 600 =0.6-1.0 culture medium, then add 2ml of fresh TSB liquid medium, culture at 37°C for 4h, and then add 200μl Escherichia coli BLT5403 (A 600=0.6-1.0)) The culture solution was cultured overnight. The next day, spread the amplified product on a 100mm LB / carb (50μg / ml) plate, incubate at 37°C for 3-4h, and use 3ml phage Extraction Buffer (20mM Tris-HCL, PH8.0; 100mM NaCl; 6mM MgSO 4 ) to elute the amplified phage, add chloroform and centrifuge, and the supernatant is the amplification product of ScFv single phage.

[0049] B. Blocking ELISA detection:

[0050] a) Determination of optimal working concentration of antigen and antibody:

[0051] In order to obtain the best antigen coating concentration and...

Embodiment 3

[0061] Example 3: Single-chain antibody sequence analysis

[0062] Use the primers on the carrier to carry out PCR amplification (the amplification method is the same as the PCR amplification method of the phage in step 5 in Example 1) to obtain S C f V , and sent them to Bao Bioengineering (Dalian) Co., Ltd. for sequencing. The upstream primers for sequencing used T7UP, and the downstream primers used T7DOWN (the primers are the primers on the T7 phage carrier in the kit). The sequencing results were analyzed, Blast was performed on NCBI, and homologous alignment was performed with the chicken antibody sequence, and their conserved sequence was found to be consistent with the chicken Ig alpha antibody sequence. The results showed that, in this study, Staphylococcus aureus was used as the antigen, and the phages panned carried chicken-derived S C f V Antibody.

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Abstract

The invention discloses a staphylococcus aureus-specific egg yolk immunoglobulin (IgY) variable region single-chain antibody. The specific egg yolk immunoglobulin variable region antibody has a nucleotide sequence shown by SEQ ID No:2 and an amino acid sequence shown by SEQ ID No:1. The single-chain antibody has relatively strong binding capacity with staphylococcus aureus, namely that the screened antibody has an obvious inhibition effect on the growth of staphylococcus aureus, and the inhibition effect and inhibition behavior are similar to those of penicillin and streptomycin, so that the single-chain antibody has a perfect application prospect in preparation of staphylococcus aureus antibody drugs or detection reagents. Moreover, the single-chain antibody has high specificity to staphylococcus aureus, and thus the laboratory early diagnosis and clinical treatment level of the staphylococcus aureus can be improved.

Description

technical field [0001] The present invention relates to chicken yolk immunoglobulin variable region single-chain antibody, in particular to a novel chicken yolk immunoglobulin variable region single-chain antibody specific to Staphylococcus aureus, and the amino acid sequence and DNA coding sequence of the variable region thereof, It also relates to the preparation method and use of the antibody. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, SA) is an important pathogen of humans and animals, belonging to the genus Staphylococcus (Staphylococcus), is a representative of Gram-positive bacteria, can cause many serious infections such as endocarditis, pneumonia, sepsis, etc. . The U.S. Centers for Disease Control reports that Staphylococcus aureus is the second-leading infection after E. coli. At present, the treatment of Staphylococcus aureus infection is mainly based on antibiotics, but their abuse leads to the frequent emergence of drug-resist...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/63C12N1/21A61K39/40G01N33/569A61P31/04C12R1/19
Inventor 徐永平李晶泉李晓宇李媛王丽丽李淑英
Owner DALIAN UNIV OF TECH
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