Artificially synthesized pig growth hormone gene and expression and purification method thereof
A technology for artificially synthesizing growth hormone, which is applied in the field of DNA recombination, and can solve the problems of insufficient expression of PGH, incapability of large-scale industrial production, and low yield.
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Embodiment 1
[0033] Embodiment 1, the artificially synthesized PGH gene of the present invention
[0034] According to the amino acid sequence of porcine growth hormone reported (http: / / www.ncbi.nlm.nih.gov / ) and the usage principle of Escherichia coli preferred codons, the present invention designs and encodes porcine growth hormone consistent with the amino acid sequence in pigs The nucleotide sequence, see the nucleotide sequence of SEQ ID NO1 in the sequence listing, its nucleotide sequence is compared with the wild-type gene sequence in the pig body (see Figure 4 ). The artificially synthesized porcine growth hormone gene is a 576bp PGH gene sequence, encoding 191 amino acids.
Embodiment 2
[0035] Embodiment 2, the highly efficient expression of artificially synthesized PGH gene in Escherichia coli
[0036] (1) Design primers with Nhe I and Xho I restriction sites: 5′-gtggt gct agc gaa gaa ttc ccg gcca-3' and 5'-gtggt ctc gag cta gaa ggc gca ggag-3′, to amplify the artificially synthesized PGH gene, and clone it into the prokaryotic expression vector pET-28a containing a strong T7 promoter+ In , the recombinant plasmid pET-28a containing the artificially synthesized PGH gene was constructed + -PGH (see Figure 5 , 6);
[0037] (2) Prepare Escherichia coli BL21 (DE3) competent cells, and use the heat shock method (42°C heat shock for 45 seconds) to recombine the recombinant plasmid pET-28a + -PGH is transformed into the host cell BL21 (DE3) to obtain an engineering strain containing the recombinant plasmid;
[0038] (3) Inoculate the engineering strain into the LB medium solution, when the engineering bacteria culture solution concentration OD 600 When it...
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