ABSTRACT
Human kallikrein-1 (hK1) gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZalphaA, then the yeast expression vector pPICZalpha-hK1 was constructed. After transformed into Pichia pastoris host X33, high-level expression transformants were screened by escalating the concentration of Zeocin (from 500 to 700 microg/mL) of YPD plate and medium. When temperature was 30 degrees C, pH 6.0 with induction duration of 64 hours in the 30 L fermenter, the highest yield can reach about 6500 u/L (1.25 g/L). The variation of glycosylation resulted in two kinds of molecules, i.e. rhK1-H with a heavy molecular weight and rhK1-L with a light one. rhK1 was purified from the supernatant through Phenyl hydrophobic interaction, Cu(2+)-charged Chelating and Anion-exchange chromatography. 0.28 g rhK1-H and 0.62 g rhK1-L can be purified from one liter supernatant. The yield recovery was 72% with a purity of > 96%. So far our yield of rhK1 is superior than known recombinant expression method reported by other researchers.
Subject(s)
Humans , Amino Acid Sequence , Base Sequence , Chromatography, Ion Exchange , Methods , Genetic Vectors , Genetics , Kidney , Metabolism , Molecular Sequence Data , Pichia , Genetics , Metabolism , Recombinant Proteins , Genetics , Tissue Kallikreins , GeneticsABSTRACT
Objective: To clone and express Treponema pallidum (TP) specific antigens P15 and P47 and use them in clinical examination. Methods: P15 and p47 genes synthesized were inserted into a GST fusion expression vector. The recombinant antigens were purified by affinity chromatography and then coated on microplates to establish an ELISA kit. Using this kit, national TP standards, sera of normal persons and patients with TP and other diseases were tested. Results: The synthesized p15 and p47 genes sequenced were identical with those of GenBank. National TP standards were tested by ELISA kit which coated with these recombinant antigens, and the results accorded with the standards. High sensitivity and specificity was showed when 2 recombinant antigens were used in ELISA to detect the sera of normal persons and patients with TP and other diseases. Conclusion: The recombinant TP specific antigen P15 and P47 are suitable for establishment of ELISA kit in clinical examination.
ABSTRACT
The leakage of glutamic-pyruvic transaminase ( GPT ) in the supernatant of primary cultures of rat hepatocytes has been used to evaluate the toxicity of the classical hepatotoxin carbon tetrachloride ( CC14 ) . The response of the isolated hepatocyte's preparation to incubate with CCl4 showed a clear dose dependency from 5 to 20 ml/ L in this system and the toxicity increased -with time of incubation. Glycyrrhizin ( 100mg/L ) and sodium caffeic acid 10 to 100 mg/L remarkably reduced GPT release in the medium of hepatocytes with CC14 while Bupleurum kunmingense polysaccharides had no significant effect at the same condition. Glycyrrhizin and caffeic acid might have protective effects against CC14. Further study is necessary. These findings also indicated that the primary cultures of rat hepatocytes is a suitable system for evaluating liver protective drugs as well as studying the function of cell metabolism.