High throughput screening atrazine chlorohydrolase mutants with enhanced activity through Haematococcus pluvialis expression system / 生物工程学报

Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.

Prokaryotic expression and polyclonal antibody preparation of nonstructural protein 2 transactivated protein of hepatitis C virus / 中华传染病杂志

Objective To express nonstructural protein 2 transactivated protein (NS2TP) of hepatitis C virus (HCV) in the prokaryotic expression system and prepare polyclonal antibody,and to study the expressions in different liver tissues.Methods NS2TP gene was amplified by polymerase chain reaction (PCR) technique and cloned into the prokaryotic expression vector pET-32a(+),which was transformed into E.coli BL21.The protein was induced by isopropyl thiogalactose (IPTG) and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting assay.The recombinant protein were expressed and purified in a large amount.The rabbit was immunized with the purified protein to prepare polyelonal antibody.The liver tissues of patients with chronic HCV infection and healthy controls were detected by immunohistochemistry method.Results The recombinant NS2TP protein (relative molecular mass:33×103 ) and polyclonal antibody with high titer and specificity were successfully prepared.NS2TP expressions in the liver of patients with chronic HCV infection were higher than those of healthy controls,and were mainly distributed in the nucleus of hepatocytes.Conclusions The NS2TP expression level and intracellular location in liver tissue of patients with chronic HCV infection are understanded,which could bring new clues for further study of the biological function of NS2TP and the pathogenesis of HCV infection.

Determination of emodin and physcion in rat plasma by nonaqueous RP-HPLC / 中成药

AIM: To determine the content of emodin and physcion in rat plasma by nonaqueous RP-HPLC. METHODS: After hydrolysis and extraction, the content of emodin in the plasma was determined by nonaqueous RP-HPLC. The separation was performed on Kromasil C 18 column (250 mm?4.6 mm, 5.0 ?m) with the mobile phase comprised of methanol-acetic acid (99.9∶0.1). The flow rate was 1.0 mL?min -1 and the detection wavelength was at 254 nm. RESULTS: The linear ranges for emodin and physcion were in the range of 0.0425-2.8 ?g?mL -1 and 0.0491-3.14 ?g?mL -1 , respectively. The average recoveries of emodin and physcion were 95.7%-100.1% and 96.2%-99.8%, with corresponding RSD of 1.3% and 1.6% respectively. CONCLUSION: This method is simple, rapid, accurate and reproducible with RP-HPLC to detect rhein in plasma.