Analysis of chemical constituents in Eupatorium lindleyanum by UPLC-Q-TOF/MS / 中草药

Objective: To analyze and identify the chemical constituents from Lindley eupatorium by using UPLC-Q-TOF/MS. Methods: The separation was performed on Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) column with gradient elution of 0.1% formic acid (A)-acetonitrile (B), the flow rate was 0.2 mL/min. The column temperature was set at 35 ℃. The MS analysis was based on information associated mode (IDA), and positive and negative ions were collected respectively. Results: A total of 26 compounds in L. eupatorium were identified by PeakView, combined with the mass spectrometry data of each chromatographic peak in the database, and the cleavage law of secondary fragment of each peak of which11 compounds were first reported for L. eupatorium. The main chemical constituents included flavonoids, nucleosides, alkaloids, phenylpropanoid, sesquiterpenoids, coumarins, polyols, etc. Conclusion: The method is accurate, reliable and effficient, which is suitable for rapid identification of ingredients in L. eupatorium, which provides a reference for clarify its efficacy and material basis.

Analysis of chemical differences between Cordyceps hawkesii and Cordyceps sinensis based on multivariate statistics / 中草药

Objective: To analyze and identify the significant different components between Cordyceps hawkesii and Cordyceps sinensis by using method of ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Methods: Mass spectrometry combined with formula finder of PeakView software and database (Human Metabolome Database, Pub Chem, Metlin) and secondary fragmentation analysis, significant different components were identified and analyzed. Results: Through OPLS-DA analysis, it was found that 12 significant different components were identified. Eleven of them were amino acids and their metabolites, and one was phosphatidylcholine. Conclusion: Surprisingly, characteristic components such as cordycepin and adenosine were not identified by significant difference analysis. In this study, it was proved that C. hawkesii can be used as a supplementary resource instead of C. sinensis, which provided scientific support for the further development and utilization of C. hawkesii.

Preparation and characterization of mono PEGylated recombinant human interferon omega / 药学学报

The amino group PEGylation of rhIFNomega with monomethoxy polyethylene glycol succinimidyl succinate (mPEG-SS, 20 000) was investigated, and the modified mixture was separated and purified by ion exchange chromatography and gel filtration chromatography. Under the optimized purification conditions, the average content ofmono PEG-rhIFNomega in the collect liquid reached 182 microg x mL(-1). The average purified yield of mono PEG-rhIFNomega exceed to 22%, and the purity of mono PEG-rhIFNomega was greater than 98% by SDS-PAGE and RP-HPLC. Relative molecular mass of mono PEG-rhIFNomega was 43 790 detected by MALDI-TOF MS. The apparent molecular mass measured by SDS-PAGE was about 60 810. The purified PEG-rhIFNomega has the characteristics of typical PEGylated protein. Activity reservation rate of mono PEG-rhIFNomega was 15.0%, while the antigenicity decreased by at least 64 folds. In addition, the acid stability, thermal stability and stability in serum and trypsin solution of mono PEG-rhIFNomega were markedly better than those of the rhIFNomega. The pharmacological properties of mono PEG-rhIFNomega were significantly improved. The prepared PEG-rhIFNomega might be developed to a novel safe and long-acting interferon.

Cloning and prokaryotic expression of major ampullate spidroin gene of spider / 生物工程学报

RT-PCR was conducted with one degenerate primer designed according to repetitive regions' amino acid sequence of major ampullate spidroin (MaSp) in spiders and adaptor primer in the SMART cDNA Library Construction Kit. By cloning and sequencing of amplified products, one cDNA clone (GenBank Accession No. AY365017) of Argiope amoena MaSp gene was obtained. The deduced amino acid sequence can be distinctly divided into two regions: (1) Repetitive region that consists of an alternating alanine-rich and glycine-rich domain in which many prolines are present; and (2) C-terminal non-repetitive region. The region coding for 272 amino acids of MaSp gene was subcloned into prokaryotic expression vector pET28b(+) and an about 26kD recombinant protein was expressed at high levels in Escherichia coli BL21 (DE3) after induction of IPTG. After being purified with metal-affinity chromatography on Ni(2+) -IDA-Sepharose columns as well as gel filtration chromatography, the recombinant protein was confirmed to be predicted MaSp by means of amino acid composition analysis and N-terminal amino acid sequence analysis. The solubility behavior of recombinant MaSp with C-terminal non-repetitive region in the present study is similar to that of recombinant dragline silk proteins without C-terminal non-repetitive region expressed by bacteria and yeast in the other studies. The result shows that absence or presence of C-terminal non-repetitive region is not a crucial factor affecting the solubility of the recombinant MaSp.

The effect of fermentation conditions on glycosylation of recombinant human interferon omega in yeast Pichia pastoris / 生物工程学报

To investigate the influence of the fermentation conditions on glycosylation of heterologous recombinant protein in yeast Pichia pastoris, the glycosylation of recombinant human interferon omega (rhIFNomega) under various fermentation conditions, e. g., cell density, initial pH, methanol concentration, duration of the induction, and medium volume were studied. The glycosylation of rhIFNomega in the continuous fermentation process under various pH values and in batch fermentation were also investigated. In 250 mL flask, the optimal cell density, initial pH, medium volume, methanol concentration and frequency of methanol induction were 250 g/L (WCW), pH6.0, less than 30 mL, 15 g/L and 3 (in every 24 h), respectively. In the continuous process, the glycosylation of rhIFNomega could be effectively improved by maintaining the pH value at 7.0-7.5. In the batch fermentation process, the expression level of glycosylated and non-glycosylated rhIFNomega were the same, but the specified value of glycosylation/non-glycosylation was significantly lower than that in the flask culture. The reason of this phenomenon will be further studied. This research lay the foundation for the scale-up of production and the enhancement of rhIFNomega glycosylation in Pichia pastoris.