关键词: 胞质分裂作用因子4, 过表达慢病毒载体, Neuro-2a细胞, 稳定转染, 过表达慢病毒

Abstract:

Objective To construct an over-expression lentiviral vector of the dedicator of cytokinesis 4 （DOCK4）， and to establish DOCK4 stably over-expressing Neuro-2a cells. Methods The DOCK4 sequence was searched in the National Center for Biotechnology Information （NCBI） and primers were designed and synthesized； polymerase chain reaction （PCR） method was used to amplify the DOCK4 gene sequences. After digestion with BamHⅠ and AgeⅠ restriction endonucleases， the DOCK4 gene sequences were ligated with the digested lentiviral vector GV492 to construct the GV492-DOCK4 over-expression recombinant plasmid. The positive clones with a similar length to the target gene fragment were screened and identified by PCR method. The GV492-control plasmid and GV492-DOCK4 over-expression recombinant plasmid were transfected into the HEK293T cells， and the lentivirus was collected and titered 48 h after transfection. The Neuro-2a cells were divided into GV492-control group and GV492-DOCK4 group， and the cells were infected with GV492-control lentivirus and GV492-DOCK4 over-expression lentivirus，respectively， and the multiplicity of infection （MOI） was 100. After 72 h of infection， the successfully infected Neuro-2a cells were screened by using puromycin （10 mg·L^-1）. The growth status of Neuro-2a cells and the expression of green fluorescent protein in various groups were observed under fluorescence microscope. Real-time quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of DOCK4 mRNA and DOCK4 protein in the Neuro-2a cells in various groups. Results The PCR results showed that the gene fragment length of the GV492-DOCK4 over-expression recombinant plasmid was approximately 691 bp. The sequencing results showed that the gene sequence of the GV492-DOCK4 over-expression recombinant plasmid was consistent with the designed over-expression sequence of DOCK4. The titers of the lentiviruses in GV492-control group and GV492-DOCK4 over-expression group were 2.5×10⁸ TU·mL^-1 and 2.5×10⁸ TU·mL^-1， respectively. The fluorescence microscope observation results showed that Neuro-2a cells in various groups grew well and expressed green fluorescent protein. The RT-qPCR results showed that compared with GV492-control group， the expression level of DOCK4 mRNA in the Neuro-2a cells in GV492-DOCK4 group was significantly increased （P<0.01）. The Western blotting results showed the specific bands near the relative molecular mass of 225 000 in various groups. Compared with GV492-control group， the expression level of DOCK4 protein in the Neuro-2a cells in GV492-DOCK4 group was significantly increased （P<0.01）. Conclusion This study successfully constructs the DOCK4 over-expression lentiviral vector and establishes the Neuro-2a cells stably over-expressing DOCK4.

Key words: Dedicator of cytokinesis 4, Over-expression lentivirus vector, Neuro-2a cell, Stable transfection, Over-expression lentivirus