Tetrodotoxin neutralizing monoclonal antibody hybridoma 5e7 and its light and heavy chain variable region genes and their application
A technology of tetrodotoxin and variable region, applied in application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0017] Example 1: Construction of anti-TTX monoclonal antibody hybridoma cell line
[0018] 1. Preparation of immune antigen: 15mg of tetrodotoxin (TTX) was dissolved in pH5.0 citrate buffer solution, this solution was slowly added dropwise to 15mgKLH / 0.01MPBS solution, while adding while stirring, adjust the pH to 7.0, add 3ml of 1% glutaraldehyde under ice bath, react at room temperature for 1.5 hours, dialysis with 0.01MPBS at 4°C for two days, change the medium several times during the period, remove denatured protein by centrifugation, and detect protein quantification. The TTX-KLH complex is immune Original, divided into 10 pieces, 1mg / piece, stored below -20℃.
[0019] 2. Preparation of antigen for detection: 10mg of Tetrodotoxin (TTX) was dissolved in pH5.0 citrate buffer, this solution was slowly added dropwise to 10mgBSA / 0.01MPBS solution, while adding while stirring, adjust the pH value At 7.0, add 3ml of 1% glutaraldehyde in an ice bath, react for 1.5 hours at room tem...
Embodiment 2
[0027] Example: Screening and identification of hybridoma cell lines with neutralizing monoclonal antibodies against TTX
[0028] 1. Determination of the LD50 of TTX's half-lethal dose for mice Choose 100 Kunming mice aged 4-6 weeks with half male and half. Dissolve TTX with double-distilled water. The concentration of the original solution is 1mg / mL and diluted with double-distilled water. 2μg / ml. After 0.5ml of TTX with a concentration of 2μg / ml was injected into the abdominal cavity of 10 Kunming mice, all the mice died within 30 minutes; 2μg / ml, 1.8μg / ml, 1.6μg / ml, 1.4μg / ml, 1.48 were used respectively. The LD50 of TTX measured at the concentration of μg / ml and 0μg / ml is 1.62μg / ml, which is 40.5μg / kg.
[0029] 2. Determination of neutralization activity of anti-TTX monoclonal antibody. Mix 0.5ml of 1mg / mL TTX monoclonal antibody (distilled water for the control group) and 1.62μg / mlTTX0.5ml into the abdominal cavity of mice. Observe the experiment and control group (10 Only) t...
Embodiment 3
[0032] Example 3 Fishing for light and heavy chain genes of 5E7 hybridoma cells with neutralizing tetrodotoxin monoclonal antibody
[0033] 1. Take 5E7 cells 5×10 of neutralizing monoclonal antibody in logarithmic growth phase 6 -10 7 After centrifugation to remove the supernatant, the cells are evenly bounced. Add 1ml TRIzol (Invitrogen) and repeatedly pipette to fully lyse the cells. After shaking for 5 minutes, add 0.2ml of chloroform, shake for 15 seconds, leave at room temperature for 2-3 minutes, 2-8℃ 12000r / min, centrifuge for 15 minutes, and take the supernatant in another In a new tube, add 500μl of isopropanol and mix well, then leave it at room temperature for 10 minutes, and centrifuge at 2-8°C at 12000r / min for 10 minutes. Wash the precipitate with 75% ethanol. After drying, dissolve the precipitate with 20μl RNase-free deionized water ( image 3 ).
[0034] 2. Take the solution containing 1μg of total RNA and add 4μl of AMV5× buffer, 0.5μl of Oligo (dT) (500ng / μl), 2...
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