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TgVP1 extracellular region antigen polypeptide, anti-TgVP1 polyclonal antibody and application of polyclonal antibody

A polyclonal antibody, antigen polypeptide technology, applied in the direction of peptide, peptide source, specific peptide, etc., can solve the problem of unclear mechanism of action

Active Publication Date: 2015-07-01
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TgVP1 contains 17 transmembrane domains, and the N-terminal has a signal peptide sequence, which can guide TgVP1 into the secretory pathway of Toxoplasma gondii, but the specific mechanism of action is still unclear

Method used

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  • TgVP1 extracellular region antigen polypeptide, anti-TgVP1 polyclonal antibody and application of polyclonal antibody
  • TgVP1 extracellular region antigen polypeptide, anti-TgVP1 polyclonal antibody and application of polyclonal antibody
  • TgVP1 extracellular region antigen polypeptide, anti-TgVP1 polyclonal antibody and application of polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of Toxoplasma gondii TgVP1 antigen polypeptide

[0020] 1. Design and synthesis of Toxoplasma gondii TgVP1 polypeptide: The Toxoplasma gondii TgVP1 protein sequence was obtained according to the Toxoplasma gondii TgVP1 sequence on GenBank (accession number: EPT25031.1), which contains 816 amino acids.

[0021] 2. The characteristics of TgVP1 protein were analyzed by DNAstar software. The analysis results are shown in Table 1. The protein has a molecular weight of 85296 Daltons and an isoelectric point of 4.83, which is an acidic protein.

[0022] Table 1

[0023] Analysis project (Analysis)

Whole Protein

Molecular Weight

85.296kDa

Length

816aa

1microgram=

11.724 pMoles

Molar Extinction coefficient

95770

1A(280)＝

1.10mg / mL

Isoelectric Point

4.83

Charge (Charge at pH 7)

-16.1

[0024] 3. Use TMHMM software to analyze the transmembrane domain of TgVP1 pro...

Embodiment 2

[0026] Example 2 Preparation of TgVP1 Antigen Polypeptide and Coupling with Carrier Protein

[0027] 1. Peptide synthesis

[0028] In order to facilitate coupling with the carrier protein, a cysteine ​​is added to the N-terminal of the antigenic polypeptide in the extracellular region of TgVP1, namely: CSSSPESEGDRFQVTA. The peptide was synthesized by Shanghai Jier Biochemical Company.

[0029] 2. Coupling of peptides and carrier proteins

[0030] a. Dilute one tube of maleimide-activated mcKLH with 200 μL ultrapure water to a 10 mg / mL solution. (Note: The above solution is blue-white and translucent, do not shake or heat, otherwise it will cause mcKLH to precipitate.)

[0031] b. Dissolving the thiol-containing hapten (ie TgVP1 antigenic polypeptide) with a coupling buffer equivalent to 1.0-2.5 times the volume of the solution in step a. For example: Dissolve 2 mg of hapten in 200-500 μL of coupling buffer and add to mcKLH. If the polypeptide is readily soluble, it can be...

Embodiment 3

[0043]Example 3 Preparation and Purification of Anti-TgVP1 Polypeptide Rabbit Polyclonal Antibody

[0044] 1. Animal immunization

[0045] The coupling product was used to immunize two New Zealand male rabbits, weighing 2-2.5kg. Freund's complete adjuvant or Freund's incomplete adjuvant was purchased from Sigma.

[0046] The immunization procedure is shown in Table 2. Dilute 100 μg of antigen with PBS to 1.25 mL, mix with an equal volume of adjuvant, vortex for 100 minutes, and check the emulsification result. Take a drop of antigen and drop it on water, and it will not dissolve and spread for one minute. Blood was taken from the ear vein before immunization, and serum was collected after standing at 4°C, and stored at -80°C as negative control serum.

[0047] Table 2 Specific immunization procedures

[0048]

[0049]

[0050] Antibody Titer ELISA Detection

[0051] (1) Experimental reagents

[0052] a. Phosphate buffered saline (PBS) (10×concentration): sodium chlo...

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Abstract

The invention discloses a toxoplasma gondii TgVP1 extracellular region antigen polypeptide, an anti-toxoplasma gondii TgVP1 polyclonal antibody and the application of the polyclonal antibody. The amino acid sequence of the toxoplasma gondii TgVP1 extracellular region antigen polypeptide is as shown in SEQ ID NO: 1 or a sequence as shown in SEQ ID NO: 1 of which the N end is connected with a cysteine. A New Zealand rabbit is immunized by the toxoplasma gondii TgVP1 extracellular region antigen polypeptide as an immunogen so that the anti-toxoplasma gondii TgVP1 polyclonal antibody can be obtained; an identification result indicates that the polyclonal antibody is capable of specifically identifying the toxoplasma gondii TgVP1 protein and can be applied to the detection of the toxoplasma gondii TgVP1 protein in tests such as ELISA, Western blot and immunofluorescence. A powerful tool is provided for the fundamental research of the protein functions and the research of the protein as a potential anti-toxoplasma gondii drug target; in short, the polyclonal antibody is wide in application prospect.

Description

technical field [0001] The invention relates to a TgVP1 extracellular region antigen polypeptide, an anti-TgVP1 polyclonal antibody and applications thereof. Background technique [0002] V-H+-PPase is a unique proton pump that exists in many animals and plants. It can hydrolyze the phosphate anhydride bond of inorganic pyrophosphate, and use the released energy to transport protons, generate an electrochemical gradient across the membrane, and play a role similar to the proton pump ATPase. V-H+-PPase can efficiently convert stored energy into H+ and / or transfer membrane gradients for a variety of different intracellular transport processes. V-PPases were first found in plants and photosynthetic bacteria, and recent studies have shown that trypanosoma, Leishmania, Toxoplasma gondii, and Plasmodium falciparum also have this enzyme. It is worth noting that this enzyme does not exist in the animal hosts of these parasites, so V-PPases have important research value as potentia...

Claims

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Application Information

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IPC IPC(8): C07K14/45C07K16/20G01N33/68G01N33/569
CPCC07K14/45C07K16/20G01N33/569G01N33/68
Inventor 罗树红郝文波肖斌
Owner SOUTHERN MEDICAL UNIVERSITY
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