An antithrombotic recombinant batroxobin lyophilized preparation
The technology of freeze-dried preparation and batroxobin is applied in the field of antithrombotic drugs, which can solve problems such as proteins with complex sugar chains in spatial structure, and achieve the effects of reducing storage and transportation costs, improving stability and enhancing safety.
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Embodiment 1
[0074] According to the Bothrops atrox genus disclosed in U.S. Genebank, the nucleotide sequence (Itoh N, Tanaka N, Mihashi S, Yamashina L, Molecular Cloning and Sequence Analysis of cDNA for Batroxobin, aThrombin-like Snake) of batroxobin (X12747) of moojeni venom species Venom Enzyme, J Biol Chem.1987 Mar5; 262 (7): 3132-5), select the genetic code of yeast preference, artificially synthesize the structural gene sequence of the site-specific transformation of batroxobin, and encode the 45th Arg The code AGA is mutated into AAG encoding Lys, and the endogenous KEX2 protease recognition sequence Arg-Arg of yeast is mutated into Arg-Lys, so KEX2 protease will not degrade the batroxobin secreted into the fermentation broth at this site, thereby making The target protein can accumulate in large quantities. In order to recombine the synthetic target gene into the yeast secretory expression vector pPICZα, an Xho I restriction site was added to the 5' end of the gene, and a stop cod...
Embodiment 2
[0077] Linearize pPICZα-Bg with DNA restriction endonuclease SacI, prepare yeast host competent and transform according to the method in Multi-Copy Pichia Expression Kit Version B of Invitrogen Company, and spread the transformed cells on YPD+500ug / ml It grows on Zeocin medium, and a single colony can appear after 3-4 days at 30°C. Clones with large and full colonies were selected for expression screening.
Embodiment 3
[0079] The high-expression engineered bacteria screened in test tubes were inoculated into shake flasks for proliferation as seed liquid, and then transferred to a 30L fermenter for pilot-scale fermentation according to the conventional method of methanol yeast fermentation. Induced for more than 200 hours. When the protein activity in the fermentation broth no longer increased, the fermentation broth was centrifuged to collect the supernatant, and the protein activity reached 84KU / ml. After microfiltration and sterilization, the active protein with a purity of more than 97% is obtained by using hydrophobic column chromatography, ion exchange column chromatography, gel column chromatography and other biochemical separation techniques.
[0080] The method for measuring the activity of similar products was used for the in vitro coagulation test. The specific test method was as follows: take 0.2ml of human-citrate anticoagulant plasma, add it to a test tube with a diameter of 1cm...
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