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Antigenic polypeptide of chlamydia pneumoniae

A technology for Chlamydia pneumoniae and antigenic polypeptides, which can be applied in the direction of fusion polypeptides, peptides, depsipeptides, etc., which can solve the problems of unclear probe specificity and difficulty in determining reaction conditions.

Inactive Publication Date: 2008-01-30
HITACHI CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The base sequence of these DNA molecules has not been determined, so the specificity of these probes is unclear
In addition, it is difficult to determine the reaction conditions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0151]Chlamydia pneumoniae Preparation of genomic DNA

[0152] Chlamydia pneumoniae The protomers were suspended in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM ethylenediaminetetraacetate (EDTA) (hereinafter referred to as "TE" buffer). To the resulting suspension, 1% sodium dodecylsulfonyl (SDS) in water and proteinase K in water (1 mg / ml) were added and the lysogens were incubated. To the resulting solution was added phenol saturated with 0.1M Tris-HCl buffer (pH 8.0). The mixture was stirred and centrifuged to recover the aqueous layer. The resulting aqueous layer was then treated with RNase and phenol / chloroform / isoamyl alcohol, followed by ethanol precipitation. got lung Chlamydia of genomic DNA.

[0153] Genomic DNA expression library preparation

[0154] Genomic DNA was digested with restriction enzymes AccI, HaeIII and AluI. Digests were treated with phenol / chloroform / isoamyl alcohol and subjected to ethanol precipitation to yield partially digested DNA. ...

Embodiment 1

[0218] Example 1: Code pair Chlamydia pneumoniae Preparation of DNA of Specific 53K Antigen Polypeptide

[0219] (A) Culture of host cells (HL cells)

[0220] In a plastic culture bottle (75cm 2 HL cells cultured close to confluence on the bottom surface of ) were rinsed with 5 ml of magnesium (-)-free phosphate-buffered saline solution (hereinafter referred to as "PBS"), and the entire plane was blocked with 5 ml of PBS containing 0.1% trypsin (w / v) , remove excess solution, incubate at 37° C. for 10 minutes, and add 5 ml of Dulbecco MEM medium containing 10% (v / v) fetal bovine serum. HL cells adhered to the inside of the flask were recovered by pipetting to obtain a cell suspension.

[0221] The cultivation in the plastic bottle was accomplished as follows: the culture flask was inoculated with 1 ml of the above-mentioned cell suspension and 5 to 20 ml of Dulbecco MEM medium containing 10% (v / v) fetal bovine serum and cultured in a 6-well plastic culture plate (vessel). ...

Embodiment 2

[0278] Example 2: with encoding containing part Chlamydia pneumoniae The preparation of the recombinant vector of the DNA of the peptide of the antigenic polypeptide, and the preparation of the transformant carrying the vector

[0279] Although the DNA obtained as described above apparently encodes a 53 kDa antigenic polypeptide, as a precaution, it was expressed as shown below to determine whether it could react with the above-mentioned antibody.

[0280] Plasmid pBBK10MM was cut with restriction enzymes BamHI and XhoI and subjected to 1.2% low melting point agarose gel electrophoresis to separate a DNA fragment of about 4.6 kbp. This fragment is purified. To each of the synthesized DNAs of SEQ ID No: 11 and SEQ ID No: 12, DNA fragments were added in an amount of 1 to 100 ng and ligated using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.). The resulting reaction product was put into Escherichia coli HB101 strain competent cells (manufactured by Takara Shuzo C...

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PUM

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Abstract

Antigen polypeptide of Chlamydia pneumoniae, which includes polypeptide A containing the sequence of at least 5 consecutive amino acid residues in the polypeptide of SEQ ID NO: 1; DNA encoding the polypeptide; a recombinant vector containing the DNA; a transformant containing the vector; Methods for producing anti-Chlamydia pneumoniae antibodies using antigenic polypeptides as antigens; methods for detecting and analyzing the anti-Chlamydia pneumoniae antibodies; applications of antigenic polypeptides; fusion proteins composed of dihydrofolate reductase and Chlamydia pneumoniae antigenic polypeptides, wherein SEQ ID The polypeptide of NO: 14 is combined with polypeptide A containing the sequence of at least 5 consecutive amino acid residues in the polypeptide of SEQ ID NO: 1; DNA encoding the fusion protein; recombinant vector containing the DNA; transformation containing the vector Methods for producing anti-Chlamydia pneumoniae antibodies using fusion proteins as antigens; methods for detecting and analyzing anti-Chlamydia pneumoniae antibodies using fusion proteins as antigens; application of fusion proteins; probes and primers for detecting and analyzing Chlamydia pneumoniae genes; application of the Methods for detecting and analyzing Chlamydia pneumoniae genes with probes or primers, and applications of the probes or primers.

Description

[0001] invention title [0002] Chlamydia pneumoniae Antigenic polypeptide, fusion protein containing the polypeptide, and its coding DNA, recombinant vector carrying the DNA, transformant containing the recombinant vector, method for producing antibody, method and reagent for detecting and / or measuring antibody, diagnosis Chlamydia pneumoniae Methods and reagents for infection, detection and / or assay Chlamydia pneumoniae Gene probes and primers and detection and / or assay Chlamydia pneumoniae Genetic Methods and Reagents. field of invention [0003] The present invention relates to Chlamydia pneumoniae Antigenic polypeptide, fusion protein containing the polypeptide, its coding DNA, recombinant vector carrying the DNA, transformant containing the recombinant vector, method for producing antibody, method and reagent for detecting or measuring antibody, diagnosis Chlamydia pneumoniae Methods and reagents for infection, detection and / or assay Chlamydia pneumoniae Genetic pro...

Claims

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Application Information

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IPC IPC(8): C07K14/295C12N15/31C12N1/21C12P21/02C12P21/08C12Q1/68G01N33/569C12N15/02A61K38/00A61K39/00C07K16/00C07K16/12C12N9/06C12N15/09C12R1/19C12R1/91
CPCC07K2319/00C12N9/003C07K14/295A61K38/00C07K2319/003A61K39/00
Inventor 井简浩小原和彦松本明
Owner HITACHI CHEM CO LTD
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