A kind of preparation method and application of cow Staphylococcus aureus alpha-hemolysin subunit vaccine
A staphylococcus, hemolysin technology, applied in vaccines, veterinary vaccines, chemical instruments and methods, etc., can solve the problems of economic losses in the dairy industry, affecting milk yield and quality, etc., achieve good cross-protection reaction, convenient application, The effect of promoting cellular immunity
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Embodiment 1
[0018] Example 1 Construction of expression vector pET28a-α-hemolysin site-directed mutation
[0019] Using the clinically isolated cow Staphylococcus aureus genome as a template, α-hemolysin was divided into two segments for PCR, the results are as follows figure 1 shown.
[0020] 1.1 The sample loading system is (50μl):
[0021]
[0022] 1.2 PCR amplification procedure:
[0023]
[0024] 1.3 Gel recovery of DNA fragments:
[0025] (1) The reaction solution in step 1.2 is subjected to 0.8% agarose gel electrophoresis (110V 30min);
[0026] (2) Under the ultraviolet light, cut the gel and recover the DNA fragments in a 1.5ml EP tube;
[0027] (3) Add 500 μl PC buffer to the 1.5ml EP tube in step (2), 50°C, water bath for 10 minutes;
[0028] (4) Move the solution in step (3) to the center of the adsorption column, let it stand for 2 minutes, and centrifuge at 12,000rpm for 30s;
[0029] (5) Discard the waste liquid, add 600μl PW buffer to the center of the adsorpti...
Embodiment 2
[0078] Example 2 Transformation of Escherichia coli BL21
[0079] Take 1 μl of plasmid and add it to 100 μl BL21 competent cells, and put it in ice bath for 30 minutes;
[0080] Heat shock at 42℃ for 90s;
[0081] Ice bath for 2 minutes;
[0082] Add 900 μl non-resistant LB culture solution in the ultra-clean bench;
[0083] Shake at 180rpm at 37°C for 1h;
[0084] Aspirate 100 μl of the bacterial solution and apply it to a Kana-resistant LB plate, and incubate overnight at 37°C.
Embodiment 3
[0085] Example 3 Large amount of induced expression
[0086] Bacteria picking: Pick a single clone into 50ml of kana-resistant LB culture medium and culture overnight at 37°C;
[0087] 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 220 rpm for 2-2.5h to OD 600 value to 0.6;
[0088] 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, and induce culture at 37°C and 220 rpm for 4 hours;
[0089] Bacteria collection: the bacteria solution was centrifuged at 6,000 rpm for 10 minutes to collect the bacteria; the bacteria were washed with 40ml PBS, centrifuged at 6,000 rpm for 10 minutes, the bacteria were collected, and stored at -20°C;
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