Prokaryotic expression, purification and identification of recombinant human atrial natriuretic peptide / 生物工程学报

In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.

Mechanisms of tumor cell’ s apoptosis triggered by gold nanorods / 中国生化药物杂志

Objective To explore the mechanism that gold nanorods trigger apoptosis in cancer cells.Methods Gold nanorods was synthesized by gold seed growing method, and its characterization was detected; gold nanorods on cell proliferation-toxicity were evaluated by CCK-8 Kit and apoptosis were detected by flow; mitochondrial membrane potential were tested by JC-1 and activation of Caspase 9 and Caspase 3 were detected by western blot. Results The results found that gold nanorods had nontoxic to normal cells, but highly toxic to tumor cells; and with the increasing of gold nanorods’ working time, the percentage of apoptotic cancer cells was increasing; in addition to, normal cells’ mitochondrial membrane potential did not change, but cancer cells had a significant reduction in mitochondrial membrane potential.Conclusion This study proves that gold nanorods induce apoptosis through the mitochondrial apoptosis pathway.

Study on the effect of small molecule inhibitors LDYS-14007 on JAK1-STAT3 signaling pathway / 中国生化药物杂志

Objective To study the effect of LDYS-14007 on JAK1-STAT3 signaling pathways.Methods MDA-MB-231 cells were treated with 10μmol,1 nmol LDYS-14007, and 10 μmol Tofacitinib,respectively.Western blot assay was used to determine the expression of JAK1,Phospho-JAK1, STAT3 and Phospho-STAT3.Results The absorbance value was linearly related to the concentration of protein C, The linear equation is A=0.0075C+0.0029, r=0.9976, The linear range of 1.08-5.08 mg/mL, With the increased concentration of LDYS-14007, the amount of Phospho-JAK1, Phospho-STAT3 were all gradually decreased.Conclusion LDYS-14007 leads to the levels of Phospho-JAK1 and Phospho-STAT3 decrease, which inhibits JAK1-STAT3 signaling pathway.LDYS-14007 may play an important role in the treatment of rheumatoid arthritis.

Construction,expression,and the analysis of antitumor activity of all anthropogenic medicinal anti-EGFR ScFv (HL-L-L) / 中国生化药物杂志

Objective To construct,express and analysis the antitumor activity of anti-EGFR ScFv(HL-L-L),and analyze bioinformatics and characterize correlation spectroscopy.Methods EGFR ScFv(HL-L-L)gene was obtained by OVER LAPING technology and construction of prokaryotic expression vector pET-28a-c(+)-VH-L-L.After induced expression,target protein HL-L-L was purified by Ni column,and its tertiary structures was predicted with SWISS - MODEL;size distribution and zeta potential distribution of the target protein HL-L-L were characterized by NanoZSP;the inhibition effects of HL-L-L on breast cancer cell proliferation were studied with the method of pharmacology.Results Prokaryotic expression vector pET-28a-c(+)-VH-L-L was constructed successfully,high purity of protein HL-L-L was obtained,and successfully predicted its three-dimensional structure;the size was about 489.7 nm,and the Zeta potential was 38.1 ,negatively charged.It had a certain inhibition effect on MCF # 7 proliferation,compared with blank group,and the difference was significant(P<0.01 ).Conclusion This study successfully express all anthropogenic medicinal anti-EGFR ScFv(HL-L-L),which has significant inhibiting effect on breast cancer cell proliferation.It provides the foun dation for further development of small molecules of anti-EGFR antibody drug.