Recombined human growth hormone gene bacilliform virus, preparation and application thereof
A technology of human growth hormone and silkworm baculovirus, applied in biochemical equipment and methods, botany equipment and methods, applications, etc., can solve the problem of loss of activity, achieve high expression efficiency and reduce production costs
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Embodiment 1
[0035] Embodiment 1. Amplification of HGH gene
[0036] A pair of primers were designed using the HGH gene as a template. The upstream primer contained an EcoR I restriction site, and the downstream primer contained a Sac I restriction site. The primers were designed as follows:
[0037] Upstream primer P1: 5'GGGAATTCAGTTTCCTAC-TATACCAC 3' (SEQ NO.2);
[0038] Downstream primer P2: 5'TTGAGCTCCTAGAAGCC-ACAGCTGCC 3' (SEQ NO.3).
[0039] Human growth hormone HGH gene was amplified by PCR from the recombinant plasmid PWR-HGH (gifted by Mr. Wu Xiangfu, Shanghai Institute of Biochemistry). Using the recombinant plasmid PWR-HGH as a template, pre-denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 1 minute; annealing at 57°C for 1 minute; extension at 72°C for 1 minute, 30 cycles; Amplify the target HGH gene fragment.
Embodiment 2
[0040] Example 2. Construction of pAcHLT-HGH vector containing HGH gene
[0041] The HGH gene fragment cut by EcoR I and Sac I was ligated by T4 DNA enzyme (MBI) and cloned into the pAcHLT-B baculovirus transfer vector (purchased from BD Biosciences Pharmingen) by EcoR I and Sac I, so that HGH The gene is placed under the control of the polyhedrin (polyhedrin, ph) gene promoter, and the recombinant transfer plasmid pAcHLT-HGH (such as figure 1 ), the gene sequence was confirmed to be correct by restriction analysis and sequencing. In the silkworm expression system, the recombinant protein HGH was expressed in the form of fusion protein.
Embodiment 3
[0042] Embodiment 3. Preparation of Bombyx mori recombinant baculovirus BmBacHGH
[0043] Take 5 μl of recombinant transfer plasmid pAcHLT-HGH DNA and 6 μl of linearized virus BmBacPAK6 DNA (purchased from Shanghai Biochemical Cell Institute), add 100 μl of serum-free TC-100 medium (purchased from GIBCOBRL Company) and mix well. Take 6 μl of Dosper (Bowringman Company) and add 100 μl of serum-free TC-100 medium and mix well. BmN cells (purchased from Shanghai Biochemical Cell Institute) previously cultured in a 35mm plate were washed twice with serum-free TC-100 medium, and the pAcHLT-HGH transfer plasmid and Dosper mixture were added dropwise, and incubated at 27°C for 4-5 On the next day, the supernatant was collected for the first round of plaque screening. Take 5 μl of the supernatant to infect the Bm N cells in a 35mm plate, discard the supernatant after 1 hour and add an equal amount of mixed TC-100 medium and low melting point agarose. After 4-5 days, plaques were pic...
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