Transgenic plant products comprising human granulocyte colony-stimulating factor and method for preparing the same
A colony-stimulating factor and granulocyte technology, applied in the fields of botanical equipment and methods, cytokines/lymphokines/interferons, biochemical equipment and methods, etc. Prospects, effects of high economic value
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Embodiment 1
[0057] Embodiment 1 Construction of chimeric gene pTZ / Phas / His / EK / hG-CSF (construct H) Amplification of hG-CSF
[0058] use Figure 16 Shown specific primers 5'GCSF-1 and 3'GCSF, amplify the coding hG-CSF mature peptide in pB / KS / hG-CSF by PCR ( Figure 15 ) nucleotide sequence (525bp). This amplification introduced a 5' NcoI site and an AccI site in the hG-CSF gene for subcloning. Prepare 50 μl of PCR reaction mixture containing: 40 ng DNA template strand of pB / KS / hG-CSF, 1XPfu buffer (Stratgene, USA), 0.2 mM dNTP, 0.5 μM 5’GCSF-1 primer, 0.5 μM 3’GCSF Primer, 2.5 units of Pfu DNA polymerase (2.5u / μl, Stratgene, USA). The PCR conditions were set as follows: 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, a total of 25 cycles; after that, 72°C for 7 min, 1 cycle.
[0059] Construction of chimeric gene pTZ / Phas / His / EK / hG-CSF (construct H)
[0060] PCR products were purified and A-tailed. Will contain 300ng PCR product, 1XTaqDNA polymerase reaction buffer (...
Embodiment 2
[0061] Construction of embodiment 2 pBK / Phas / SP / His / EK / hG-CSF (construct SH)
[0062] The construction of the pGEM(R)-T / hG-CSF plasmid was described above. Then, use NcoI and NotI enzymes to excise the target gene from the vector and clone it into the pET / SP / His / EK vector containing part of phaseolin signal peptide sequence, histidine tag and EK site, thus forming the plasmid pET / SP / His / EK / hG-CSF. Cut the entire gene cassette with NdeI and AccI enzymes and clone it into the pBK / Phas / SP vector containing the promoter and other parts of the lentin signal peptide sequence. The resulting vector is named pBK / Phas / SP / His / EK / hG -CSF( Figure 12 ).
Embodiment 3
[0063] Construction of embodiment 3 chimeric gene pBK / Phas / SP / hG-CSF (construct S)
[0064] first use Figure 16The two specific primers shown, 5'GCSF-2 and 3'GCSF, amplify the nucleotide sequence (525bp) encoding the mature peptide of hG-CSF in the pB / KS / hG-CSF vector by PCR. This amplification introduces a 5'NdeI site and a 3'AccI site in the gene of interest for subcloning. PCR reaction mixture solution (except that 5'GCSF-1 primer is replaced with 5'GCSF-2 primer) and PCR condition are identical with embodiment 1. The PCR product was purified and subjected to A-tailing reaction as in Example 2. The A-tailed PCR product was ligated into pGEM(R)-T vector. Use NdeI and AccI enzymes to excise the target gene from the pGEM(R)-T / hG-CSF-NoHis vector and clone it into the pBK / Phas / SP vector containing phaseolin promoter, phaseolin signal peptide sequence and terminator . The resulting plasmid was named pBK / Phas / SP / hG-CSF ( Figure 13 ).
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