RESUMO
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.
Assuntos
Humanos , Sequência de Aminoácidos , Sequência de Bases , Cromatografia por Troca Iônica , Métodos , Vetores Genéticos , Genética , Rim , Metabolismo , Dados de Sequência Molecular , Pichia , Genética , Metabolismo , Proteínas Recombinantes , Genética , Calicreínas Teciduais , GenéticaRESUMO
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.