Preparation and application of anti-staphylococcus aureus polyclonal antibody
A polyclonal antibody, staphylococcus technology, applied in the fields of bioengineering and immunology, can solve problems such as unsatisfactory effect and accelerated drug-resistant Staphylococcus aureus
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Embodiment 1
[0017] Construction of embodiment 1 expression vector pET28a-mntc
[0018] Using the clinically isolated cow Staphylococcus aureus genome as a template, the PCR results are as follows: figure 1 As shown, the nucleotide sequence of the obtained MntC gene is shown in SEQ ID NO.2.
[0019] 1.1 The sample loading system is (50μl):
[0020]
[0021]
[0022] 1.2 PCR amplification procedure:
[0023]
[0024] 1.3 Double digestion reaction (40μl system):
[0025]
[0026] Into a 1.5ml EP tube, add sample and mix according to step 1.3, and then place the two 40μl reaction solutions in a constant temperature water bath at 37°C for 2h.
[0027] 1.4 Gel recovery of DNA fragments:
[0028] (1) Perform 0.8% agarose gel electrophoresis (110 V for 30 min) on 40 μl of the system reaction solution in step 1.3;
[0029] (2) Under the ultraviolet light, cut the gel and recover the DNA fragments in a 1.5ml EP tube;
[0030] (3) Add 500 μl PC buffer to the 1.5ml EP tube in step (...
Embodiment 2
[0064] Example 2 Transformation of Escherichia coli BL21
[0065] Take 1 μl of plasmid and add it to 100 μl BL21 competent cells, and put it in ice bath for 30 minutes;
[0066] Heat shock at 42℃ for 90s;
[0067] Ice bath for 2 minutes;
[0068] Add 900 μl non-resistant LB culture solution in the ultra-clean bench;
[0069] Shake at 180rpm at 37°C for 1h;
[0070] Aspirate 100 μl of the bacterial solution and apply it to a Kana-resistant LB plate, and incubate overnight at 37°C.
Embodiment 3
[0071] Example 3 Large amount of induced expression
[0072] Bacteria picking: Pick a single clone into 50ml of kana-resistant LB culture medium and culture overnight at 37°C;
[0073] Transfer: transfer the bacterial solution to 500ml of kana-resistant LB culture medium at a ratio of 1:100, shake 3.5L in total, culture at 37°C and 220rpm for 2-2.5h to OD 600 value to 0.6;
[0074] Induction: bacterial solution OD 600 After the value reaches 0.6, add 500 μl IPTG (1M) to a final concentration of IPTG of 1 mmol / L, induce culture at 37°C and 220 rpm for 4 hours;
[0075] Bacteria collection: the bacteria solution was centrifuged at 6,000rpm for 10min to collect the bacteria; the bacteria were washed with 40ml PBS, centrifuged at 6,000rpm for 10min, the bacteria were collected, and stored at -20°C;
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