Keratin BD-2, preparation method, pharmaceutical composition and application thereof
A technology for BD-2 and keratin, which is applied in the field of keratin BD-2, its preparation method and its pharmaceutical compositions and uses, to achieve the effects of analgesic effect, strong effect and reducing the number of writhings
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Embodiment 1
[0114] Example 1 shake flask fermentation preparation protein BD-2 crude solution A (TB medium)
[0115] Synthesize the nucleotide sequence shown in SEQ ID No.2, and transfer it into the pET-28a (+) vector; sequencing confirms that the expression vector containing the correct sequence is obtained; the expression vector is transfected into BL21 (DE3) cells, The expression-competent host cells containing the target nucleotide sequence are obtained. Add it to LB medium, culture in a shaker at 37° C. and 220 rpm for 1 hour to obtain a recombinant strain.
[0116] The recombinant strain was dipped and streaked on the LBA plate containing Kanamycin, and the plate was placed upside down in a constant temperature incubator at 37°C for overnight cultivation for 16 hours.
[0117] Prepare 400ml of TB culture medium, divide into 2 bottles, each bottle is 200ml. Add Kanamycin (final concentration 50 μg / ml) to each bottle (200ml) of TB medium, take a single colony on the plate and add it...
Embodiment 2
[0124] Example 2 shake flask fermentation preparation protein BD-2 crude solution B (other medium)
[0125] Synthesized and sequenced in Example 1 to obtain an expression vector containing the sequence shown in SEQ ID No.2; the expression vector was transfected into BL21 (DE3) cells to obtain expression-competent host cells containing the target nucleotide sequence.
[0126] Prepare 20 ml of LB medium, take 800 μl, add 50 μl of host cells containing the target coding sequence, and culture in a shaker at 37° C. and 220 rpm for 1 hour.
[0127] Dip the above bacterial solution and streak it on the LBA plate containing Kanamycin, and place the plate upside down in a constant temperature incubator at 37°C for overnight cultivation for 16 hours.
[0128] Take 10ml of LB medium, add Kanamycin (final concentration 50 μg / ml), take a single colony on the plate and add it to LB medium, in a shaker, under the conditions of 37°C and 220rpm overnight amplification culture for 15 hours, to ...
Embodiment 3
[0134] Example 3 fermenter preparation protein BD-2 crude solution C
[0135] Synthesized and sequenced in Example 1 to obtain an expression vector containing the sequence shown in SEQ ID No.2; the expression vector was transfected into BL21 (DE3) cells to obtain expression-competent host cells containing the target nucleotide sequence. Add it to LB medium, culture in a shaker at 37° C. and 220 rpm for 1 hour to obtain a recombinant strain.
[0136] On the LBA plate containing Kanamycin, add 100 μl of the recombinant strain, spread it with a spreader until it dries evenly, and place the plate upside down in a constant temperature incubator at 37°C for overnight culture. Take three single colonies and streak them on a plate containing Kanamycin, culture the plate overnight, and after three batches of shake flask fermentation and expression verification confirm that it is correct, preserve the strain with 15% glycerol, and pack it into 0.8ml each to obtain Working cell bank, fr...
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