Murine anti-MPT32 protein hybridoma cell strain 13B12, monoclonal antibody based on mouse anti-MPT32 protein hybridoma cell strain 13B12 and application of monoclonal antibody
A technology of hybridoma cell line and monoclonal antibody, which is applied in the direction of anti-bacterial immunoglobulin, anti-animal/human immunoglobulin, application, etc., and can solve the problems of low detection sensitivity, low specificity, complicated operation, etc. , to achieve the effect of high detection sensitivity, simple operation and high specificity
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Embodiment 1
[0030] Example 1 Screening of mouse anti-MPT32 protein hybridoma cell lines
[0031] (1) Preparation and purification of MPT32 protein
[0032] The recombinant plasmid containing the MPT32 gene fragment was transformed into the host Escherichia coli (8Rosetta (DE3) bacteria of the BL21 series) to obtain the recombinant bacteria, and the recombinant bacteria were expanded by using the LB liquid medium containing kanamycin resistance. The volume ratio was 1:100, and the bacterial solution was transferred to the above-mentioned medium for induction expression. When the OD value of the bacterial solution was between 0.4 and 0.6, isopropyl thiogalactoside (IPTG) was added to make the final concentration of IPTG as 1mM, cultured at 30°C for 4 hours, centrifuged, and the cells were collected. The cells with a wet weight of about 2 g were added to 30 mL of PBS buffer for resuspending, and then placed in an ice-water mixture for sonication (power of 400 W, ultrasonic for 3 s, interval...
Embodiment 2
[0038] Example 2 Preparation of mouse anti-MPT32 protein monoclonal antibody
[0039] (1) Preparation of ascites fluid and purification of MPT32 protein monoclonal antibody
[0040] Hybridoma cells were washed with sterile PBS solution at 5 × 10 6 The cells were injected intraperitoneally into liquid paraffin presensitized mice. After 7 to 10 days, the ascites was collected, centrifuged at 3000 rpm for 10 min at room temperature, and the supernatant was collected. The antibody in ascites was crudely purified by the ammonium octanoate sulfate method. The crudely purified antibody was further purified by AKTA protein purification system and 1ml Protein G purification prepacked column according to the purification manual provided by GE. The purified antibody was used for subsequent antibody detection and functional experiments.
[0041] The electrophoresis results after antibody purification are as follows: figure 1 shown. Depend on figure 1 It can be seen that after inject...
Embodiment 3
[0045] Example 3 Gene verification of mouse anti-MPT32 protein monoclonal antibody
[0046] In this example, the total RNA extracted from the hybridoma cell line 13B12 was used as a template, and the variable region genes of the heavy chain and light chain of the monoclonal antibody expressed by the hybridoma cells were obtained by reverse transcription, PCR amplification and sequencing. The gene of the variable region is spliced with the gene of the constant region of the murine antibody in the database to obtain the complete expression sequence of the recombinant monoclonal antibody.
[0047] The entire experimental process is divided into three steps: cell culture, cDNA preparation, and antibody variable region gene amplification and sequencing analysis. 13B12 was cultured; (2) the total RNA in the cell samples was extracted, and the cDNA was obtained by reverse transcription using the tailing method; (3) the heavy and light chains were amplified by polymerase chain react...
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