ABSTRACT
PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
Subject(s)
Animals , Humans , Baculoviridae , Genetic Vectors , Recombinant Proteins , Sf9 Cells , SpodopteraABSTRACT
Objective To observe the influence of Zhibai Qianqing(ZBQQ) Decoction combined with Levofloxacin on sperm quality of chronic prostatitis(CP) patients and explore its possible mechanism through sperm apoptosis rate.Methods Fifty eight cases were divided into 2 groups randomly.Twenty eight cases were served with ZBQQ decoction combined with Levofloxacin(observe group),30 cases were treated by Levofloxacin tablets(control group).The curative effect of the two groups was compared one month later.All the cases underwent semen analysis.Sperm apoptosis rates were measured by TUNEL before and after the treatment.Results Sperm liquefaction time of CP group was significantly longer than the normal group(P0.05).The clinic cure rate of observe group was 35.71%,and overall effective rate was 85.71%,significantly higher than that of control group(P