The Initial Establishment of a New Poxviral/Lentiviral Hybrid System for Efficient Lentiviral Vector Production / 生物化学与生物物理进展

Efficient gene delivery and sustained gene expression are required for successful human gene therapy.Although viral vectors are considered the most efficient vehicles for gene transfer,currently available viral vectors have not fully achieved these two requirements.Lentiviral vectors(LVs)can integrate into host chromosomes,allowing long-term gene expression,in addition,these vectors are non-toxic and minimally immunogenic since no viral genes are encoded in the vector genome,but are still limited to in vitro or ex vivo gene delivery because of their relatively low titers using transient transfection experiments.In order to develop an efficient transient transfection method for large-scale production of high titer lentiviral vector stocks,a minimal lentiviral vector producing system based on vaccinia virus that synthesizes T7 RNA polymerase was developed.BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA,the packaging plasmid pGAGPOL and the envelope plasmid pVSVG,and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000TM.After 4 days,the culture supernatant of lentiviral vectors was collected,the RNA from the supernatant was examined by the RT-PCR,the protein from the supernatant was examined by Western blot,and the supernatant was used to transfect normal 293T,HepG2 and Vero,which were observed by the immunofluorescence microscopy.The type of cell lines,plasmids dosage and the MOI(the proportion between cell numbers and virus copies)were considered so critical to the output of this system that 3?3?3 factorial design was used to explore the yield optimization of this system.As judged by the results of RT-PCR and the Western blot,lentiviral vectors were found in the culture supernatant;as judged by immunofluorescence with microscopy,293T,HepG2 and Vero which were transfected by the supnantant expressed the report protein-green fluorescent protein(GFP),the results confirmed the valid infectivity of the lentiviral vector produced by the system.Eventually,the best titers of lentiviral vector stocks was up to 1.3?108 tu/ml,which is one order of magnitude higher than the output of classical manufacture system.The new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established.It provides the basis for the future development of industrial application.

Comparison of the immunochromatographic malaria P. f/P. v test with microscopic examination and PCR for diagnosis of malaria / 中华检验医学杂志

100 parasites/?l, 72.73% with

Screening and identification of anti-HRPII single chain Fv antibodies of Plasmodium falciparum / 细胞与分子免疫学杂志

To develop simple, rapid, and efficacious diagnostic methods for malaria is one of the remaining key tasks for malaria control. Previously, we have created a phage-displayed antibody library against Plasmodium falciparum. Six clones of antibody with good reactivity to HRP-II in ELISA were isolated from the library after 3 rounds of enrichment. Soluble ScFvs were produced and the characteristics were determined. The results of Western blot showed that they could bind to HRP-II specifically and had a relative molecular mass(Mr) about 31 000. The work provided a solid fund for diagnostic kit development for malaria.

Expression and Immunocompetence Characterization of Plasmodium falciparum Lactate Dehydrogenase / 中国寄生虫学与寄生虫病杂志

Objective To express lactate dehydrogenase (LDH) gene of Plasmodium falciparum FCC1/HN in the E. coli TG1 and analyse its immunocompetence. Methods The LDH gene of the P. falciparum was specifically amplified by polymerase chain reaction, and the recovered gene fragment was cloned into pGEX 4T 1 vector for expression of fusion protein with glutathione S transferase(GST). The recombinant plasmid was transformed into the E. coli TG1. Four mice (Kunming strain) were immunized with purified expressed protein(antigen) and the polyclonal antibodies were collected. The immunocompetence of recombinant protein was analysed by ELISA and Western blot. Results The LDH gene of P. falciparum was successfully expressed in the E. coli TG1. The expressed protein exhibited a specific reaction with immune sera obtained from rabbits immunized with P. falciparum . The specific humoral responses were induced in mice and the titer of the specific antibody was 1∶16 by two dimensional diffusion assay. Conclusion The LDH gene of P. falciparum has been successfully expressed in the E. coli TG1 and the expressed protein has high antigencity.

Soluble Expression of Plasmodium falciparum Glutamate Dehydrogenase in Escherichia coli, and its Purification and Identification / 中国寄生虫学与寄生虫病杂志

Objective To make soluble expression of Plasmodium falciparum (FCC1/HN) glutamate dehydrogenase(GDH) in Escherichia coli, purification and immunocompetence identification of the recombinant non-fusion GDH. Methods The GDH gene was cloned into prokaryotic expression vector pET23(a) to form recombinant expression vector pET23(a)/GDH. pET23(a)/GDH was transformed into E.coli BL21(DE3). Induced by IPTG(isopropyl-beta D-thiogalactoside), GDH was highly expressed in the supernatant after sonication. The soluble recombinant GDH was purified by Source-Q and Source-S chromatography. Enzyme-linked immunosorbent assay and Western blotting were carried out to identify the immunocompetence of the purified product. Results SDS-PAGE analysis showed that the soluble GDH protein accounted for approximately 15% of the total bacterial protein. By two-step ion-exchange chromatography, the purity of GDH reached more than 90% and the GDH possessed high antigenicity. Conclusion The soluble expression of GDH results in an integral three-dimensional structure epitope with high biological activity.