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Conjugates and their preparation methods and applications

A technology of conjugates and markers, which is applied in the field of conjugates and their preparation, and can solve problems affecting the activity of antigens

Active Publication Date: 2018-12-28
FAPON BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In immunoassay, in order to improve the sensitivity, the higher the labeling ratio, the better, but the higher the labeling ratio will affect the antigen activity

Method used

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  • Conjugates and their preparation methods and applications
  • Conjugates and their preparation methods and applications
  • Conjugates and their preparation methods and applications

Examples

Experimental program
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Effect test

preparation example Construction

[0061] In addition, the present invention also provides figure 1 The preparation method of the above-mentioned conjugate shown comprises the following steps:

[0062] S110, providing a gene expression vector for expressing a fusion protein, the fusion protein includes a syphilis detection antigen and an auxiliary protein chimerized at the N-terminal of the syphilis detection antigen.

[0063] Generally, the construction process of a gene expression vector is as follows: select a gene fragment of an auxiliary protein, design primers with restriction enzyme sites, amplify the auxiliary protein gene fragment by PCR, and connect it to the expression vector treated with the corresponding restriction enzymes. , the recombinant plasmid was obtained. Select a gene fragment of the syphilis detection antigen, design primers with enzyme cutting sites, PCR amplify the syphilis detection antigen gene fragment, and connect it into the recombinant plasmid cut with the corresponding enzyme t...

Embodiment 1

[0105] Embodiment 1, preparation of coated antigen recombinant plasmid

[0106] Select the DNA segment corresponding to 63aa-156aa of Treponema pallidum 17Kda (TpN17) gene (GeneBank serial number M74825), and design primers. The sequence of the upstream primer is shown in SEQ ID No.3, and the sequence of the downstream primer is shown in SEQ ID No.4 . The upstream primer has a BamHI site, the downstream primer has an EcoRI site, and the coding sequence of 6 His amino acids before the EcoRI site and the stop codon TAA.

[0107] For PCR amplification, the PCR conditions were: denaturation at 94°C for 5 minutes; (94°C, 30s, 55°C, 30s, 72°C, 30s)×30 cycles; finally, extension at 72°C for 10 minutes. Reclaim the fragment of PCR (molecular biology extraction and recovery kit used in the present invention are all purchased from Shanghai Huashun Bioengineering Co., Ltd.), then digest with BamHI and EcoRI (various molecular biology enzymes used in the present invention Both were purc...

Embodiment 2

[0108] Example 2, expression and purification of coated antigen

[0109] Transform the pET-24a-TP17 plasmid into BL21(DE3) competent cells (Tiangen Biochemical Technology Co., Ltd., Cat. No. CB105), and spread it on a medium containing 100 μg / mL kanamycin sulfate (Shanghai Sangon Bioengineering Technology Service Co., Ltd., Hereinafter referred to as Sangon (hereinafter referred to as Sangon, product number KB0286), cultivate overnight at 37°C, pick a single clone, and use the same concentration of kanamycin sulfate (Shanghai Sangon Bioengineering Technology Service Co., Ltd., hereinafter referred to as Sangon, product number KB0286) ) in 500mL LB medium at 37°C with shaking until OD600=1.0, and induced with IPTG (Sanko, product number IB0168) at a final concentration of 0.5mM for 4 hours at 37°C. 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 liqui...

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Abstract

The invention discloses a conjugate. The conjugate comprises fusion protein and a label, wherein the fusion protein comprises a syphilis detection antigen and accessory protein embedded in the N terminal of the syphilis detection antigen; the label is labeled on the accessory protein. According to the conjugate, the accessory protein is embedded in the N terminal of the syphilis detection antigen, and the label is labeled on the accessory protein, so that the direct action between the label and the antigen is avoided; compared with the conventional reagent, the conjugate has the following advantages: while the labeling proportion improves, the antigen is not wrapped by the label, so that the reduced antigen activity caused by the combination of antigenic determinant and the label is avoided, and the antigen activity is not influenced; during detection, the proportion of the label can be appropriately improved, so as to improve the sensitivity and specificity of detection. The invention also discloses a 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 is a chronic systemic infectious disease, the pathogen is Treponema pallidum, also known as Treponema pallidum (TP), mainly through sexual contact and blood transmission. Syphilis can produce a variety of symptoms and signs, which appear and disappear from time to time, and the course of the disease can last for a long time. It can almost invade all organs of the body. It is a sexually transmitted disease that seriously endangers the health of our people after AIDS. In recent years, the incidence of syphilis has been on the rise in my country. According to the 2014 syphilis key epidemic report, syphilis has ranked third in the number of reported incidences of Class B infectious diseases since 2009, especially those who are positive for syphilis antibodies screened in prenatal clinics. The num...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/13G01N33/68G01N33/571
Inventor 范凌云孟媛李泓彦
Owner FAPON BIOTECH INC
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