Conjugate, preparation method and application thereof

A technology of conjugates and markers, applied in the field of conjugates and their preparation, can solve the problem of reduced antigen activity

Active Publication Date: 2015-10-28
GUANGDONG FAPON BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the active site of the antigen in the detection reagent is easily wrapped by the label,

Method used

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  • Conjugate, preparation method and application thereof
  • Conjugate, preparation method and application thereof
  • Conjugate, preparation method and application thereof

Examples

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preparation example Construction

[0058] In addition, the present invention also provides the preparation method of the above-mentioned conjugate, such as figure 1 shown, including the following steps:

[0059] S110. Providing a gene expression sequence for expressing a mutant syphilis detection antigen, wherein the mutant syphilis detection antigen is obtained by mutating 1 or 2 lysines in the middle of Treponema pallidum antigen.

[0060] Specifically, the mutant syphilis detection antigen is obtained by mutating one or two lysines in the middle of the Treponema pallidum antigen into glycine or other neutral amino acids.

[0061] Generally, the gene expression sequence can select the corresponding gene sequence from the gene bank (GeneBank) according to the antigenic protein to be expressed, and design mutation primers as needed to point-mutate one or a certain nucleotide sequence.

[0062] In one embodiment, the gene expression sequence comprises:

[0063] (a), the nucleotide sequence shown in SEQ ID No.1...

Embodiment 1

[0104] Example 1 Preparation of Coated Antigen Recombinant Plasmid

[0105] The DNA segment corresponding to 59aa-102aa of Treponema pallidum 15Kda (TpN15) gene (GeneBank No. U73115.1) was selected, and primers were designed. The sequence of the TpN15 upstream primer is shown in SEQ ID No.7, and the TpN15 upstream primer has a BamHI site. The sequence of the TpN15 downstream primer is shown in SEQ ID No.8, and the TpN15 downstream primer has an EcoRI site.

[0106] PCR amplification, PCR conditions are: denaturation at 94°C for 5min; 30 cycles at 94°C for 30sec, 57°C for 30sec, 72°C for 20sec, and finally 72°C for 10min to obtain the DNA fragment shown in the sequence of SEQ ID No.2. Reclaim the fragment of PCR (AxyPrep DNA gel recovery kit, American Axygen, item number: AP-GX-50), then digest with BamHI and EcoRI (various molecular biology enzymes used in the present invention are all purchased from Dalian Bao Bioengineering Co., Ltd.), reclaim the digested product, and con...

Embodiment 2

[0107] Example 2 Expression and purification of coated antigen

[0108] The pET-24a-TPN15A plasmid was transformed into BL21(DE3) competent cells (Tiangen Biochemical Technology Co., Ltd., product number CB105), and coated with 100 μg / mL kanamycin sulfate (Shanghai Sangon Bioengineering Technology Service Co., Ltd., A506636) on the LB plate, cultivated overnight at 37°C, picked a single clone, cultured with shaking at 37°C in 500mL LB medium containing the same concentration of kanamycin sulfate to about OD600=1.0, and used a final concentration of 0.5mM IPTG ( Sanko, Cat. No. A100487) were induced at 37°C for 4 hours. Collect the cells by centrifugation at 5000g for 20 minutes at 4°C, resuspend the cells in 20ml lysis buffer (50mM Tirs-HCl, pH8.0, 1mM EDTA, 100mM NaCl) per liter of bacterial liquid, sonicate, and centrifuge at 12000g at 4°C After 20 minutes, after identification by SDS-PAGE electrophoresis, most of the target protein was distributed in the supernatant of the...

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Abstract

The invention discloses a conjugate, which includes a mutant syphilis detection antigen and a marker. Through gene mutation of the syphilis detection antigen, one or more lysine in the middle of the syphilis detection antigen can be mutated. The lysine easy to combine with the marker in the middle of the syphilis detection antigen turns into other amino acids, so that the marker is marked at a lysine site at the N terminal or C terminal of the mutant syphilis detection antigen and does not combine with the middle part amino acid, thus avoiding wrapping of the antigen active site by the marker. Compared with traditional reagents, the antigen active site of the conjugate is not wrapped by the marker, the antigenic activity decrease caused by combination of antigen determinants and the marker can be avoided, thereby not affecting the antigen activity. The conjugate can improve the detection sensitivity and specificity when used for syphilis detection, and can avoid the prozone phenomenon. The invention also discloses the preparation method and application of the conjugate.

Description

technical field [0001] The invention relates to the field of immunoassay, in particular to a conjugate and its preparation method and application. Background technique [0002] Syphilis (syphilis), also known as "mildew sore", "wide sore", "cotton sore" or "red bayberry sore", is a chronic systemic infectious disease and is listed as one of the three major chronic infectious diseases in the world. The pathogen of syphilis is Treponema pallidum, also known as Treponema pallidum (TP), which was first discovered in 1905 by Hoffmann and Xie Wending in Germany. Syphilis is mainly transmitted through sexual contact and blood, and if a woman is infected with syphilis during pregnancy, it will be transmitted to the fetus through the placenta, resulting in congenital syphilis. Syphilis can produce a variety of symptoms and signs. It invades the genitals and skin in the early stage, and invades all organs of the body in the late stage, especially the cardiovascular and central nervou...

Claims

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Application Information

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IPC IPC(8): G01N33/571
CPCG01N33/571
Inventor 孟媛李瑞净卢盛萍刘莉莉
Owner GUANGDONG FAPON BIOTECH CO LTD
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