Preparation technology of recombination human blood vessel inhibitor K1-3 and its application in medicine for treating tumour
An angiostatin and anti-tumor drug technology, applied in the field of genetic engineering, can solve the problem of not being able to obtain long-term treatment of angiostatin protein, and achieve the effect of inhibiting the proliferation of human umbilical vein endothelial cells
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Embodiment 1
[0060] Construction of prokaryotic expression vector pHB
[0061] 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 characteristics of high-efficiency expression of the loaded cloned gene in Escherichia coli, and its structure is shown in figure 1 .
Embodiment 2
[0063] Cloning and Expression of Human Angiostatin Gene
[0064]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:
[0065] 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
[0069] Purification, denaturation and renaturation of angiostatin
[0070] (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) After washing 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.
[0071] (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.
[0072] (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 c...
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