ELISA Quantitative Detection Method of Exogenous EPSPS Protein in Plants
A quantitative detection method and protein content technology, which is applied in the field of ELISA quantitative detection of exogenous EPSPS proteins in plants, can solve problems such as expression level detection, and achieve the effect of simple and effective operation and simple and easy-to-obtain consumables
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Embodiment 1
[0052] Antigen preparation
[0053] 1. Construction of prokaryotic expression vector
[0054] Design primers according to the sequence of the target gene (5-enolpyruvylshikimate-3-phosphate synthase) disclosed in CN 103834674 A patent, the upstream and downstream primers are respectively shown in SEQ ID NO: 2 and SEQ ID NO: 3 , the EPSPS protein sequence is shown in SEQ ID NO:1. The target fragment was amplified by PCR, and ligated into the pET28a vector after digestion to obtain a recombinant expression vector.
[0055] 2. Small test expression
[0056] 1) Plasmid transformation and strain preservation: Transform the recombinant vector into Escherichia coli BL21(B)E.coli BL21 OrigamiB, spread on LB solid medium containing 50mg / L antibiotic Kan, and culture at 37°C for 12-16h. Pick a single colony and culture it in LB liquid medium containing 50 mg / L Kan for 12-16 hours, and store the glycerol bacteria at -80°C.
[0057] 2) Strain activation: Take 100 μL of transformed Esc...
Embodiment 2
[0069] Antibody preparation
[0070] 1. Animal immunity
[0071] Two 2-month-old female New Zealand white rabbits were used for immunization, and the antigen prepared in Example 1 was used for three times of immunization, and multi-point injections were taken in the thigh muscles. For each immunization, the amount of antigenic protein for each New Zealand white rabbit was 500 μg, emulsified with an equal amount of Freund's complete adjuvant for the first immunization, emulsified with an equal amount of Freund's incomplete adjuvant for the second immunization, and emulsified with an equal amount of Freund's incomplete adjuvant for the third immunization. Physiological saline was mixed and injected into the ear vein. The specific immunization procedures are shown in Table 1.
[0072] Table 1 is the immunization scheme for polyclonal antibody preparation
[0073]
[0074] Blood was collected 14 days after the second immunization, and the antibody titer was measured by an in...
Embodiment 3
[0085] Drawing of standard curve
[0086] 1. Selection of the concentration range of the standard curve
[0087] In order to ensure the accuracy of the experimental results, the range of the standard curve is particularly important. Only when the standard curve is within an appropriate range can it be conducive to the construction of the standard curve and finally calculate the results accurately according to the standard curve. The R of the standard curve 2 To be greater than 0.98, the OD450 value of each scattered point should be between 2-0.08 to avoid exceeding the optimum reading range of the instrument.
[0088] If the concentration of the standard curve is too high, it is easy to make a misjudgment during the qualitative process. It is impossible to construct a good standard curve beyond the optimum degree range of the instrument, and the calculated result is also small, even lower than 0, such as Figure 5 In A, set the concentration to 1000ng / ml, 750ng / ml, 500ng / ml, ...
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