ABSTRACT
[Abstract ] Objective The purpose of this study was to construct a short hairpin RNA (shRNA) interference lentiviral vector targeting the humanβ-COP gene and to evaluate its inhibitory effect on β-COP in THP-1 cells. Methods We designed and synthesized 4 humanβ-COP-specific oligonucleotide sequences and inserted them into the pGMLV-SC1 vector to construct a recombinant vector fol-lowed by transfection of HEK 293T cells with the recombinant vector and Lenti-HG Mix to produce lentiviruses and detect the viral con-tent.After infecting the THP-1 cells with the packaged lentiviruses , we analyzed the inhibitory effect of β-COP-shRNA on the β-COP gene by quantitative PCR and Western blot . Results Sequencing confirmed that the β-COP-specific oligonucleotide sequences were in-serted into the lentiviral vector and the lentiviruses were packaged in the transfected HEK 293T cells, with the final viral content of 1 × 109 TU/mL.Quantitative PCR showed that the 4 β-COP-shRNA vectors significantly decreased the mRNA expression of β-COP (P<0.01), with interference rates of 16.9 %,32.5%, 74.0%, and 50.3%, respectively.Western blot also indicated their inhibitory effect on the protein expression of β-COP, with an inhibition rate of 76.4% onβ-COP-shRNA3. Conclusion Lentiviral shRNA interference vectors targeting human β-COP were constructed successfully , which could suppress the expression of the human β-COP gene.
ABSTRACT
Objective To explore the best technology to produce recombinant mouse endostatin by thioredoxin fusion expression system.Methods The recombinant plasmid pThioHis-endo was further transformed into different E.coli.,including BL21,JM109,DH5?.After induction with IPTG of different concentration or for different time period,thioredoxin-endo fusion protein was expressed in E.coli.and the product was identified by SDS-PAGE.The recombinant endostatin expression and extraction,wash,degeneration and refolding of inclusion bodies were carried out by the optimized route,and the product was further purified by affinity chromatography through Ni~(+) column and identified by SDS-PAGE.Results No difference of endostatin expression in BL21,JM109,DH5? was found.The optimal concentration of IPTG was 0.9 mmol/L and optimal inductive phase was 5 h.The soluble recombinant endostatin could be obtained by affinity chromatography through Ni~(+) column.Conclusion Soluble endostatin recombinant fusion protein of high purity and yield could be obtained by the optimized technique route.
ABSTRACT
Objective To explore the effects of recombinant expression plasmid of human glucagon-like peptide-1 (hGLP-1) analog gene (2?Val2-hGLP-1) on blood glucose, serum insulin level and pancreatic island in diabetic rats. Methods The diabetic rat model was reproduced by intraperitoneal injection of streptozotocin. The rats were then divided into 3 groups randomly (8 each): recombinant plasmid pIRES2-EGFP/Val2-hGLP-1 transfection group, empty plasmid pIRES2-EGFP transfection group, and diabetic rat model control group. Moreover, 8 untreated SD rats were set as normal control. Each rat in empty plasmid transfection group and recombinant plasmid transfection group was injected via tail vein with 110?g plasmid, whibe those in diabetic model control group and normal control group were treated with equal volume of normal saline solution. Blood glucose and serum insulin levels of rats were determined 30 days after experiment, and glucose tolerance test and insulin tolerance test were performed to estimate insulin sensitivity. The pathological changes in pancreatic island and insulin secretion were evaluated with HE and immunohistochemistry staining, respectively. Results Compare with normal group, diabetic model group and empty plasmid transfection group, the blood glucose level significantly lowered (P0.05). Meanwhile, the insulin secretion was increased and the pathological changes in pancreatic islands were alleviated in recombinant plasmid transfection group compared with that in diabetic model control group. Conclusions hGLP-1 analog gene transfection may be able to promote the proliferation of pancreatic islands and enhance sensitivity to insulin, thus significantly lower blood glucose level and ameliorate the lesion of pancreatic islets in diabetic rats.