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
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[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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