Construction method and application technology of rolGLP-HV engineering bacterium
A technology of engineering strains and Escherichia coli is applied in the field of biopharmaceuticals to delay and improve thrombosis symptoms
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Embodiment 1
[0036] Construction of a fusion gene rolGLP-HV. Including the following steps:
[0037] (1) Using pMD-GLP as a template (the gene sequence of rolGLP-1 has been applied for a national invention patent by the inventor, and the patent publication number is: CN 101824388A), obtain the rolGLP-1 gene fragment by PCR (using Pfu enzyme). The PCR primers used in the experiment were designed according to the sequence of rolGLP-1:
[0038] P1: 5′-CATTCTGAGGGTACTTTCACTTCTG-3′
[0039] P2: 5′-TCTACCATCAACCAACCAAGCGATG-3′
[0040] (2) Using gene semi-synthesis method to obtain rHV 1 Gene fragment( image 3 A). First according to rHV 1 Synthesize four oligonucleotide fragments from the gene sequence: P3, P4, P5, P6:
[0041] P3: 5′-GTTGTCTACACTGACTGTACTGAATCTGGTCAAAACTTGTGTTTGTGTGAAGGTTCCAACGTTTGTGGTCAAGGTAACAAGTGTATCTT-3′
[0042] P4: 5′-TTACTGGTGAGGGTACTCCTAAGCCACAAATCCCATAACGACGGTGACTTCGAGGAGATTCCAGAGGAATACTTGCAATAAAAGCTTGGG-3′
[0043] P5: 5′-GGCTTAGGAGTACCCTCACCAGTAACACATTGGTTT...
Embodiment 2
[0053] Construction of an Escherichia coli expression vector pNK-EC-rolGLP-HV containing rolGLP-HV fusion gene.
[0054] The rolGLP-HV gene fragment was digested with HindIII. Plasmid pET-22b(+) was digested with Nco I, blunted with Pfu enzyme, and then the large fragment of the vector was digested with HindIII. The digested vector and gene fragments were ligated ( figure 2 ). Picking positive transformants is the expression vector used in the present invention, the Escherichia coli expression vector pNK-EC-rolGLP-HV ( Figure 5 ).
Embodiment 3
[0056] Induced expression of Escherichia coli engineering strain NK-rolGLP-HV-E007 and purification of rolGLP-HV.
[0057] Select the NK-rolGLP-HV-E007 strain and culture it in a shaker flask at 37°C and 230r to OD 600 When the final concentration was 0.6-0.8, add the inducer IPTG with a final concentration of 1mmol / L, induce at 25°C for 12h, centrifuge the bacterial cells for ultrasonic disruption, and extract the protein for Tricine-SDS electrophoresis detection to prove the successful expression of rolGLP-HV( Figure 6 A). The supernatant after ultrasonic treatment was purified by affinity chromatography (Ni-NTA chromatography column) to obtain high-purity rolGLP-HV ( Figure 6 B). Western blot results ( Figure 6 C) shows that the constructed Escherichia coli has successfully expressed rolGLP-HV.
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