Monoclonal antibody for detecting necrobacter leukotoxin, kit and application thereof
A monoclonal antibody and leukotoxin technology, applied in biological testing, anti-bacterial immunoglobulin, measuring devices, etc., can solve the problems of poor repeatability and low accuracy of test results, and achieve high coincidence rate, strong repeatability, The results are accurate
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0034] In the present invention, the preparation method of the Bacillus necrosis leukotoxin ALKTA2 recombinant protein preferably includes the following steps: using the whole genome DNA of Bacillus necrosis as a template, amplifying the ALKTA2 fragment by PCR, connecting the cloning vector pMD-18T vector, and obtaining the recombinant clone pMD- 18T-ALKTA2 vector; the recombinant cloning vector was transformed into competent cells, cultured, and the bacterial solution was obtained; the plasmid was extracted from the bacterial solution, and the obtained plasmid pMD-18T-ALKTA2 and expression vector pGEX-6p-1 were double-enzyme digested and merged. After purification, the purified ALKTA2 and pGEX-6p-1 were connected, and conventionally transformed and cultured; 600nm When it reaches 0.6 to 0.8, inductive culture is performed to obtain the induced bacterial liquid; the bacterial liquid is ultrasonically broken to obtain the ALKTA2 protein expressed in the form of inclusion bodies;...
Embodiment 1
[0060] Establishment of hybridoma cell lines
[0061] 1. Antigen Preparation
[0062] Sequencing and identification were carried out by Harbin Boshi Biotechnology Company. The specific steps are as follows:
[0063] Necrobacterium leukotoxin ALKTA2 recombinant protein (purified Necrobacterium leukotoxin ALKTA2 recombinant protein) was obtained by the following method:
[0064] The whole genome DNA of Bacillus necrosis was extracted using a bacterial genome extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd.
[0065] Using the whole genome DNA of Bacillus necrosis as a template, PCR-amplified the ALKTA2 fragment to obtain the amplified product, and the amplified product was purified by Tiangen Genome Purification Kit to obtain the purified product of the target gene;
[0066] ALKTA2 primer sequences are: ALKTA2 U: 5'GAAggatccAAAGAAACTTATAACACTC 3' (SEQ ID No.3, upstream primer); ALKTA2 L: 5'GCGctcgagAAAATAAGTTCGTTAGCTTA3' (SEQ ID No.4, downstream primer); ...
Embodiment 2
[0108] Monoclonal antibody (ascites) preparation
[0109] Ascites was prepared from ALKTA 2E9-positive hybridoma cells, and 8-week-old female BALB / c mice were injected intraperitoneally with 300 μL of incomplete Freund's adjuvant 3 days in advance to collect positive hybridoma cells with good growth conditions. The number of cells is about 25cm 2 60% to 70% of the bottom of the cell bottle, discard the cell culture supernatant, add 1640 medium without double antibody and serum to mix the cells, the injection volume is 400 to 500 μL per mouse, observe the abdomen of the mouse in time after injection, about 7 days, when the abdomen When the swollen, slightly erected coat, the abdomen was sterilized with alcohol cotton balls, and a 5mL disposable syringe was used to absorb the pale yellow ascites, centrifuged at 5000rpm / min for 5min, and the clear liquid was absorbed as ascites, that is, monoclonal antibodies; the ascites was frozen at -80 ℃.
PUM
| Property | Measurement | Unit |
|---|---|---|
| concentration | aaaaa | aaaaa |
| concentration | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More