A kind of β-lactam drug receptor protein and its application
A technology of receptor protein and lactam, which is applied in the field of β-lactam drug receptor protein, can solve the problems of immunological detection and analysis methods that are difficult to meet the requirements and poor sensitivity of penicillins
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Embodiment 1
[0076] This embodiment is a method for preparing a β-lactam drug receptor protein whose amino acid sequence is shown in SEQ ID NO: 1. The preparation method includes the following steps:
[0077] According to the amino acid sequence shown in SEQ ID NO: 1, the corresponding gene sequence shown in SEQ ID NO: 5 was obtained through bioinformatics screening;
[0078] The sequence of SEQ ID NO: 5 was amplified by PCR. The specific reaction conditions were: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, and 30 cycles, followed by extension for 5 minutes at 4°C terminate the reaction;
[0079] The PCR amplified product and pET28a were subjected to double enzyme digestion, and then the digested products were mixed, and T4 DNA ligase was added at 4°C overnight, and then the ligated product was transformed into the cloning host DH5α competent cells, and The positive recombinant plasmid was n...
Embodiment 2
[0084]The present embodiment is the preparation method of the horseradish peroxidase-labeled ampicillin, and the preparation method comprises the following steps:
[0085] (1) Dissolve 40 mg of ampicillin in 4 ml of 0.05M pH7.4 phosphate buffer, then put 5 mg of N-hydroxysuccinimide and 5 mg of dicyclohexylcarbodiimide into the solution of ampicillin, at room temperature , Reaction under stirring conditions for 4h;
[0086] (2) Add 4ml of horseradish peroxidase solution with a concentration of 5mg / ml to the reacted solution, then add 1mg of carbodiimide, and react at room temperature for 2h under stirring conditions;
[0087] (3) After the reaction, refrigerate overnight and dialyze for 24 hours using a dialysis bag with a molecular weight cut-off of 10 kDa to obtain horseradish peroxidase-labeled ampicillin.
Embodiment 3
[0089] This embodiment is a method for preparing an ELISA plate coated with the amino acid sequence prepared in Example 1 as the receptor protein shown in SED ID No: 1. The preparation method includes the following steps:
[0090] Dilute the receptor protein whose amino acid sequence is SED ID No: 1 with pH9.6 carbonate buffer to 0.5 μg / ml, and add 100 μL of amino acid sequence to each well of the microplate plate as SED ID No: 1 The indicated receptor protein dilutions were incubated overnight at 4°C; then, the receptor protein dilutions with the amino acid sequence of SED ID No: 1 were spin-dried, and the plate was washed with phosphate Tween buffer 3 Next, add 1% gelatin solution to the microtiter plate, incubate at 37°C for 2h, and then dry the 1% gelatin solution to obtain the coated plate. The amino acid sequence prepared in Example 1 is SED ID No: 1 Microtiter plates of the indicated receptor proteins.
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