Abstract:

Objective To construct the over-expressed lentivirus vector of PRDM5 gene and establish the Neuro-2a cells stably transfected PRDM5， and to provide the basis evidence for exploring the effect of PRDM5 in pathogenesis of ischemic stroke（IS）. Methods The sequence of PRDM5 was searched and designed based on NCBI. The PRDM5 gene was amplified by PCR and ligated with the lentiviral vector GV492 digested by BamHⅠ and AgeⅠ restriction enzymes to form the GV492-PRDM5 over-expression recombinant plasmid. The positive clones with similar length and size to the target gene fragment were screened by PCR and sent to Shenggong Bioengineering （Shanghai） Co. Ltd. for identification. The correctly-sequenced GV492-control plasmid and GV492-PRDM5 over-expression recombinant plasmid were transfected into the HEK293T cells， respectively. After 48 h of transfection， the lentiviruses were collected by centrifugation， and they were GV492-control lentivirus and GV492-PRDMS over-expression lentivirus； the titers of these two lentiviruses were determined by lentiviral titer assay. The Neuro-2a cells were divided into GV492-control group and GV492-PRDM5 group， and then infected with GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus， respectively， with a lentivirus multiplicity of infection （MOI） of 100. The Neuro-2a cells successfully infected with GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus were screened with puromycin （10 mg·L^-1） after 72 h of infection. The growth status and the expression of green fluorescence protein of Neuro-2a cells in GV492-control group and GV492-PRDM5 group were observed by fluorescence microscope. The expression levels of PRDM5 mRNA and PRDM5 protein in the Neuro-2a cells in two groups were detected by real-time fluorescence quantitative RCR（RT-qPCR） and Western blotting methods. Results The PCR results showed that the length of the positive transformant of GV492-PRDM5 recombinant plasmid was about 684 bp， and the gene sequence of GV492-PRDM5 over-expression recombinant plasmid was consistent with the designed and synthesized PRDM5 over-expression sequence. The titers of GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus were both 2.5×10⁸ TU·mL^-1. The Neuro-2a cells in GV492-control group and GV492-PRDM5 group grew well， and the expressions of green fluorescence protein were found under fluorescence microscope.The RT-qPCR results showed that the expression level of PRDM5 mRNA in the Neuro-2a cells in GV492-PRDM5 group was significantly increased compared with GV492-control group（P<0.01）. The Western blotting results showed that the specific bands appeared in the Neuro-2a cells in GV492-control group and GV492-PRDM5 group with a relative molecular weight of 75 000； compared with GV492-control group， the expression level of PRDM5 protein in the Neuro-2a cells in GV492-PRDM5 group was increased（P<0.01）. Conclusion The over-expression lentivirus vector of PRDM5 gene is successfully constructed， and the stably transfected GV492-PRDM5-Neuro-2a cells are established.

Key words: PRDI-BF1 and RIZ domain proteins, Over-expression lentivirus vector, Stable expression, Neuro-2a cells