A kind of hybridoma cell line secreting Mycobacterium tuberculosis esat6 protein specific antibody, its antibody and application
A hybridoma cell line and antibody technology, applied in anti-bacterial immunoglobulin, application, immunoglobulin and other directions, can solve the problems of false positive test results, poor accuracy, high specificity, etc., and achieve simple and good operation. Sensitivity and specificity, good specificity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0053] The aforementioned preparation method of the anti-ESAT6 antibody may include the following steps: cultivating the aforementioned antibody expression system under conditions suitable for expressing the aforementioned antibody, thereby expressing the aforementioned antibody, and purifying and isolating the aforementioned antibody.
[0054] Appropriate conditions for expressing the antibody should be known to those skilled in the art. Those skilled in the art can select a suitable medium based on experience and culture under conditions suitable for the growth of host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isol...
Embodiment 1
[0103] The acquisition of embodiment 1 hybridoma cell strain
[0104] Obtaining the hybridoma cell line whose deposit number is CCTCC NO: C2019287.
[0105] 1. Expression, purification and identification of immunogen rHis-CFP10-ESAT6 protein
[0106] The recombinant strain pET-30a-CE / BL21(DE3) was revived for protein expression, purification and identification. The bacterial pET-30a-CE / BL21(DE3) (constructed and preserved in the laboratory) was taken from glycerol and placed on a kanamycin LB (Kan / LB) plate, cultured at 37°C for 16-24h. Pick a single colony and culture it overnight in a constant temperature shaker at 37°C, inoculate it in fresh Kan / LB liquid medium at a ratio of 1:100, and culture it at 37°C for 2-3 hours until the bacterial liquid OD 600 When the value reaches 0.4-0.6, add IPTG (final concentration: 0.5mmol / L) and place in low temperature and low speed (30°C, 180rpm) to induce expression for 5-6h. Centrifuge the induced bacterial solution and discard the s...
Embodiment 2
[0125] Example 2 Preparation and Purification of Anti-ESAT6 Monoclonal Antibody
[0126] 1. Preparation of Ascites
[0127]The method of inducing ascites in vivo was carried out according to the conventional method. Inject 0.3-0.5mL of liquid paraffin intraperitoneally into female BALB / c mice over 10 weeks old. After 7-10 days, the hybridoma cells in the logarithmic growth phase are washed with sterile PBS and resuspended in sterile PBS. Inject the cells resuspended in sterile PBS into the peritoneal cavity of BALB / c mice, 5×10 5 Each / 0.2mL / only. The state of the mice was observed 7-10 days after the immunization, and the ascites was collected when the abdomen was obviously enlarged and the movement was inconvenient. Centrifuge at 3500rpm for 10min, draw the supernatant, detect the ascites titer by indirect ELISA, aliquot and store at -70°C. The monoclonal antibody secreted by the hybridoma cell line with the deposit number CCTCC NO: C2019287 or its subcultured cell line (...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More