Activating effect of fish NKEF-A (Natural Killer Enhancer Factor-A) recombinant protein on adaptive humoral immunity and application of recombinant protein
A technology of NKEF-A and recombinant protein, which is applied in the direction of peptide/protein components, medical preparations containing active ingredients, blood diseases, etc., can solve unclear problems and achieve high safety, small dosage and high efficiency Effect
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Embodiment 1
[0020] Embodiment 1, cDNA molecular cloning of encoding NKEF-A protein
[0021] According to the instructions of the Trizol kit (TaKaRa), RNA was extracted from the spleen of puffer fish, and the RNA was reverse-transcribed into cDNA using the 3'-Full RACE Core Set kit (TaKaRa). Using the cDNA as a template, the forward primer F1: 5'- CCATGGCTGCAGGCAAAGCTC-3' and reverse primer R1: 5'-CTCGAGGTGCTTGGAGAAGAACTC-3' were amplified by PCR (see Table 1 for the system) to obtain the coding nucleotide sequence of NKEF-A recombinant protein. The PCR conditions were: 94°C pre-denaturation for 4 minutes, 94°C denaturation for 30 seconds, 56°C annealing for 30 seconds, 72°C extension for 30 seconds, and 72°C extension for 10 minutes, a total of 35 cycles. The PCR products were identified by 1.2% agarose gel electrophoresis, and the results showed that The NKEF-A cDNA band that matched the expectation was found ( figure 1 ).
[0022] The amplified NKEF-A cDNA fragment was recovered using...
Embodiment 2
[0029] Embodiment 2, NKEF-A recombinant protein prokaryotic expression plasmid construction
[0030]Using the NcoI and XhoI restriction sites respectively introduced in the PCR primers described in Example 1, the pT / NKEF-A recombinant plasmid was digested, and the digested fragment was identified by 1.2% agarose gel electrophoresis, and recovered by the method of Example 1 The target fragment was recombined into the expression plasmid pET28(a) cut by NcoI and XhoI, transformed into E.coli DH5α, and spread on the LB plate containing Kan (kanamycin, final concentration 50μg / ml) , cultivated overnight at 37°C, screened recombinants, identified by PCR and enzyme digestion, and obtained the prokaryotic expression plasmid of NKEF-A, which was named pET28(a) / NKEF-A.
Embodiment 3
[0031] Embodiment 3, NKEF-A recombinant protein preparation
[0032] The prokaryotic expression plasmid pET28(a) / NKEF-A obtained in Example 2 was transferred into Rosetta (BL21) engineering bacteria, and after recombinant screening, positive clones were picked and inoculated in 5ml containing Kan (final concentration 50 μg / ml) In the LB liquid medium, put it on a shaker at 37°C, and cultivate it at 250rpm for 5h, then add the culture solution to the LB liquid medium containing Kan (final concentration 50μg / ml) at a volume ratio of 1:100, and expand the culture at 37°C , cultivated at 250rpm to bacterial concentration OD 600 When it is between 0.6 and 1.0, add IPTG with a final concentration of 0.1 mM, then transfer to a low-temperature shaker at 20°C, and culture overnight at 100 rpm to induce the expression of soluble recombinant protein. The next day, collect the bacterial liquid, centrifuge at 5000g for 15 minutes, discard the supernatant, collect the bacterial cells, resu...
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