Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objecti...Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objective protein. Methods： By polymerase chain reaction （PCR） technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK＋/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1（＋）. After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Results： The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion： SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.展开更多
In the present study, a chitosan-coated calcium phosphate nanoparticle（CS/CaP/siRNA NP） was developed to deliver si RNA for treatment of cervical cancer. The CS/CaP/siRNA NPs were prepared by the nano-precipitation ...In the present study, a chitosan-coated calcium phosphate nanoparticle（CS/CaP/siRNA NP） was developed to deliver si RNA for treatment of cervical cancer. The CS/CaP/siRNA NPs were prepared by the nano-precipitation method. The resulted NPs had a uniform spherical morphology with a size of ～194 nm and a zeta potential of ～＋27 mV. In vitro experiments demonstrated that these NPs could efficiently deliver si EGFR into Hela cells and significantly down-regulate the EGFR expression level, which was probably associated with enhanced cell adhesion of chitosan, leading to extended residence time of cell internalization. Then the internalized CS/CaP/siRNA NPs exhibited pH-responsive disassembly of NPs, resulting in the enhanced release of si RNA and rapid lysosomal escape into cytoplasm. Moreover, in vivo anticancer results showed that the CS/CaP/siRNA NPs had significant inhibitory effects on tumor growth after intratumoral injection in Hela tumor xenografted nude mice, accompanying with no obvious changes of body weight during the whole experimental period. All these results indicated that the CS/CaP/siRNA NPs would have great potential to deliver si RNA for the treatment of cervical cancer via mucosal administration.展开更多
基金the Scientific Research Foundation of Education Committee of Hunan (07C713 and 07C708)
文摘Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objective protein. Methods： By polymerase chain reaction （PCR） technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK＋/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1（＋）. After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Results： The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion： SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.
基金National Natural Science Foundation of China(Grant No.81473158,81690264 and 81773650)the New Drug R&D program of China(Grant No.2018ZX09721003-004)the Opening Project of Key Laboratory of Drug Targeting and Drug Delivery System,Ministry of Education(Sichuan University)
文摘In the present study, a chitosan-coated calcium phosphate nanoparticle（CS/CaP/siRNA NP） was developed to deliver si RNA for treatment of cervical cancer. The CS/CaP/siRNA NPs were prepared by the nano-precipitation method. The resulted NPs had a uniform spherical morphology with a size of ～194 nm and a zeta potential of ～＋27 mV. In vitro experiments demonstrated that these NPs could efficiently deliver si EGFR into Hela cells and significantly down-regulate the EGFR expression level, which was probably associated with enhanced cell adhesion of chitosan, leading to extended residence time of cell internalization. Then the internalized CS/CaP/siRNA NPs exhibited pH-responsive disassembly of NPs, resulting in the enhanced release of si RNA and rapid lysosomal escape into cytoplasm. Moreover, in vivo anticancer results showed that the CS/CaP/siRNA NPs had significant inhibitory effects on tumor growth after intratumoral injection in Hela tumor xenografted nude mice, accompanying with no obvious changes of body weight during the whole experimental period. All these results indicated that the CS/CaP/siRNA NPs would have great potential to deliver si RNA for the treatment of cervical cancer via mucosal administration.
