Preparation method and application of porcine TLR4 polyclonal antibody

A polyclonal antibody and protein technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve problems such as restricting research

Active Publication Date: 2016-09-07
GUANGXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of studying the interaction between porcine bacterial and viral diseases and TLR4 (porcine Toll-like receptor 4, Toll-like receptors 4), it is necessary to use various experimental methods such as Western-blot, IFA, Co-IP and FCM, All these research methods do not require anti-porcine TLR4 polyclonal antibody as a support, and the lack of porcine-derived antibodies in the biological market seriously restricts the research on porcine TLR4

Method used

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  • Preparation method and application of porcine TLR4 polyclonal antibody
  • Preparation method and application of porcine TLR4 polyclonal antibody
  • Preparation method and application of porcine TLR4 polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Construction of TLR4 prokaryotic expression vector and genetically engineered bacteria.

[0019] According to the amino acid sequence analysis of Sus scrofa toll-like receptor 4 (TLR4) mRNA (accession number: KF460453.1) in GenBank, its hydrophobicity and linear epitope, the prokaryotic expression gene sequence was determined to be 2043-2052 with a total length of 510bp. The amino acid sequences are shown in SEQ.ID.No.3 and SEQ.ID.No.1 in the S sequence table respectively, and the clone primer F1 / R1 and the prokaryotic expression primer F2 / R2 of the target gene are designed. Collect PK-15 cells infected with swine fever virus, extract the total RNA of the cells and reverse transcription, and use primers F1 / R1 to amplify the large TLR4-KZ fragment (621bp) ( figure 1 ), and its nucleotide sequence is shown in SEQ.ID.No.2 in the Sequence Listing. Using the TLR4-KZ plasmid as a template, the prokaryotic expression sequence TLR4 (170) was obtained by PCR amplification...

Embodiment 2

[0023] Example 2 Obtaining and Purifying Recombinant TLR4 Protein

[0024] The positive genetically engineered bacteria were inoculated into sterile LB medium containing ampicillin, cultured at 37°C at 200 rpm until the optical density value (OD600) of the bacterial solution reached 0.6, IPTG was added to the final concentration of 0.05mM, and the expression was induced at 30°C for 6h, 4°C The cells were collected by centrifugation at 6000 rpm for 20 minutes, the cells were resuspended in lysis buffer, and the cells were lysed by ultrasonic wave, and the cells were centrifuged at 6000 rpm for 30 minutes at 4° C. to collect the supernatant and the precipitate. After the pellet was resuspended in PBS, and the supernatant were added 5× loading buffer and boiled for 5 minutes. SDS-PAGE was used to detect the expression level and expression form of the target protein ( image 3 ).

[0025] The recombinant protein obtained in the present invention is expressed in the form of inclusion bo...

Embodiment 3

[0026] Example 3 Preparation and Obtaining of Polyclonal Antibodies

[0027] Eight 4-week-old Kunming mice were immunized with multiple subcutaneous injections on the back on days 1, 7, 21, and 35. For the first immunization, the recombinant protein is mixed and emulsified with the same amount of Freund's complete adjuvant, and the protein immunity of each mouse is about 500 μg. Subsequent immunization was emulsified with an equal amount of Freund's incomplete adjuvant and recombinant protein, and the protein immunization amount for each mouse was about 300 μg. On the 7th day after the three immunizations, randomly select a mouse with tail-cutting and collect 100-200μL of blood into an EP tube, let it stand at room temperature for 2 hours, centrifuge at 12,000 rpm for 10 minutes at 4°C, separate the serum, and detect the prepared polyclonal by Western-blot The reactivity of antibody serum with recombinant protein confirms that mice produce specific antibodies, and the dilution o...

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Abstract

The invention discloses a preparation method of a porcine TLR4 polyclonal antibody. According to the preparation method, recombinant TLR4 protein (antigen) and freund's adjuvant are emulsified, then an immune animal obtains polyclonal antibody serum, wherein the recombinant TLR4 protein has the amino acid sequence as indicated in the sequence table SEQ. ID. No. 1. It is shown through tests that the antibody prepared through the method is good in reactivity and specificity; due to the fact that a TLR4 gene is a member of a significant pattern recognition receptor Toll-like receptor family targeted to innate immune response, the porcine TLR4 polyclonal antibody can be applied to detection of the porcine TLR4 protein and related research; a good foundation is laid for researching the interaction mechanism between porcine bacterial and viral infection diseases and TLR4 and the virus immunity escape mechanism, and a new target is provided for developing new generation vaccine and medicine. Meanwhile, a prokaryotic expression vector constructed through the method is high in recombinant protein expression efficiency, and the separation and purification method is simple and easy to operate.

Description

Technical field [0001] The invention belongs to the technical field of polyclonal antibody preparation, and in particular relates to a preparation method and application of a porcine TLR4 polyclonal antibody. Background technique [0002] TLR4 is a type I transmembrane protein, an extremely important member of the TLR family, and the main receptor for the innate immune system to recognize pathogenic microorganisms. TLR4 can recognize lipopolysaccharide of Gram-negative bacteria and play an important role in the occurrence of bacterial infectious diseases. In recent years, more and more studies have found that TLR4 is also widely involved in the occurrence of viral infectious diseases and the immune escape of viruses. TLR4 is the only receptor in the TLR family that can use four linker molecules (MyD88, MAL / TIRAP, TRIF and TRAM) to transmit cascade signals. Through downstream IRAK and TAF3 and TRAF6, it activates transcriptional regulators, such as NF-κB, AP-1 and a series of in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/06A61K39/42A61P31/14
CPCC07K16/28
Inventor 罗廷荣赵倩楠李晓宁李晓泉应雪陶倩姜炳星
Owner GUANGXI UNIV
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