Modification, expression and bioactivity analysis of hK-Fc fusion protein / 生物工程学报

To prolong serum half-life of human kallikrein (hK) and enhance its secretion rate, we modified hK gene and constructed a new form of recombinant hK protein (hK'-Fc). We amplified hK gene and Fc sequence, replaced the signal peptide of hK gene with murine signal peptide, constructed native expression plasmid of pcDNA-hK-Fc and modified expression plasmid of pcDNA-hK'-Fc, then transfected to CHO cells respectively. After the stable cell lines were screened, we compared the secretion rate between native fusion protein and modified fusion protein, purified fusion protein through Protein A+G affinity chromatography column and investigated the bioactivity of fusion protein. The results showed that recombinant vectors encoding fusion protein hK-Fc and hK'-Fc were constructed successfully; CHO cell lines stably secreting fusion protein were obtained, the yield is higher than 11 mg/L; Secretion rate was enhanced by 5-10 times after the signal peptide of fusion protein was modified; Fusion protein has enzymatic activity in vitro. The above results could promote the following researches on serum half-life of the fusion protein and develop a new stroke medicine with better clinical efficacy.

The effects of atorvastatin on protein kinase C and C-reactive protein in experimental atherosclerosis / 中华急诊医学杂志

Objective To reveal the protective effects of atorvastatin against atherosclerosis independent of cholesterol-lowering effect, we investigated the effects of atorvastation on the expression of protein kinase C (PKC) and C-reactive protein in experimental atherosclerosis of rats.Method Fifty female Sprague-Dawley rats were randomly divided into normal diet group (n = 10, control group), vitamin D3 injection and high cholesterol diet group (n = 40). After 8 weeks, vitamin D3 injection and high cholesterol diet rats were randomized to receive either atorvastatin (5 mg. kg-1. d-1) (n = 20, atorvastatin group) or normal diet (n = 20, model group). Another eight weeks later, all rats were killed and part of their aortas were examined by light and electron microscope and the left were removed for western blot analysis to measure PKC; At the begin and end of experiment, serumcollected for lipid and C-reactive protein determining determination.Results Cholesterol, low-density lipoprotein, triglyceride levels in atorvastatin group were significantly lower than those in model group but higher than control group. The pathologic changes in atorvastatin group were less severe than those in model group, there showed no any pathological changes in control group. The levels of C-reactive protein in model group[(18.64 ± 0.94) mg/L] were higher than those in control group [(9.21 ± 0.21)mg/L] (P<0.05). C-reactive protein levels also differed significantly between control and atorvastatin group (12.52 ± 0.65 mg/L)( P<0.05). PKC levels were significantly higher in model group (7786.12 ± 264.75)and atorvastatin group (4267.57 ± 233.94) than in control group (2468.75 ± 145.53)(all P<0.05). But compared with model group, PKC levels were markedly lower in the atorvastatin group ( P<0.01 ).Conclusions Atorvastatin may be useful not only as a cholesterol-lowering agents but also as anti-arteriosclerotic agent that provide vascular protection by inhibition PKC expression and inflammatory reaction.