Preparation technology of recombination human blood vessel inhibitor K1-3 and its application in medicine for treating tumour
An angiostatin and preparation process technology, applied in the field of genetic engineering, can solve the problem of inability to obtain angiostatin protein for long-term treatment, and achieve the effect of inhibiting the proliferation of human umbilical vein endothelial cells
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Embodiment 1
[0059] Construction of prokaryotic expression vector pHB
[0060] The pET-3a plasmid (product of Novagen, USA) was digested with restriction endonucleases BglII and AatII to obtain a DNA fragment of 719 bases. This fragment contains the T7 promoter, terminator, ribosome binding sequence and NdeI / BamHI subcloning site. This fragment was inserted into the pUC19 plasmid (product of BRL, USA) to replace the BamHI / AatII restriction fragment in the pUC19 plasmid. Thus, a new plasmid was obtained, called pHB, which has the characteristic of enabling high-efficiency expression of the loaded cloned gene in Escherichia coli, and its structure is shown in FIG. 1 .
Embodiment 2
[0062] Cloning and Expression of Human Angiostatin Gene
[0063] The preparation process of the present invention is mainly a product obtained through processes such as gene cloning, bacterial fermentation culture, extraction, and purification. The steps are as follows:
[0064]1. Obtain the 1-3 cDNA of the human angiostatin gene, ie, the plasminogen Kringle structure, from the human fetal liver cell cDNA library by PCR method screening. First, two primers were prepared: 5'-GGGAATTCCATATGAACGTATATCTATCGGAGTGTAAGACTGGGAATGG-3' and 5'-CTAGTCTAGATTAGGAGTGACTGGACGCTATCTTACAGTACT--3'. Using 1 μl of human fetal liver cell cDNA as a template, add 2 μl of each of the above two primers at a concentration of 10 nM per ml. 8 μl 2.5 mM dNTP, 5 μl 10×PCR buffer, 1 μl Taq DNA polymerase. The PCR conditions were: 94°C, 5 minutes; 94°C, 45 seconds; 58°C, 45 seconds; 72°C, 1 minute, for 30 cycles, and then extended at 72°C for 10 minutes. The human angiostatin K1-3 gene fragment was separate...
Embodiment 3
[0068] Purification, denaturation and renaturation of angiostatin
[0069] (1) After ultrasonically disrupting the genetically engineered bacterial cells expressing human angiostatin, centrifuge at 7000G for 15 minutes, discard the supernatant, and use bacterial cell washing solution (Tris-HCl 50mM, EDTA 0.01M, NaCl 100mM, pH8.0) Wash twice, add 0.5% Triton X-100 cell washing solution, ultrasonically lyse the cells at 4°C, and centrifuge at 2500-5000G three times to wash the inclusion bodies obtained.
[0070] (2) Add inclusion body dissolving solution (Tris-HCl 50mM, EDTA 1mM, NaCl 100mM, 40mM DTT, 8M urea, pH 8.0) to the inclusion body precipitation to fully dissolve the inclusion body.
[0071] (3) Add double the amount of carbonate buffer (NaHCO 3 -NaOH 50mM, EDTA 0.01M, pH11, use NaCl to adjust the conductivity to 6ms / cm), slowly adjust the mixed solution to pH9 with 1N NaOH, pass through a G-25 gel filtration column after degassing, remove the denaturant, and collect th...
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