Optimization of electroporation parameters for ctenopharyngodon idellus kidney cells and transient expression of grass carp reovirus NS26 protein.
- Author:
Yan LI
;
Ya-Nan ZHANG
;
Li-Qun LV
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Line;
Cyprinidae;
Electroporation;
Fish Diseases;
virology;
Gene Expression;
Kidney;
virology;
Reoviridae;
genetics;
physiology;
Reoviridae Infections;
veterinary;
virology;
Viral Nonstructural Proteins;
genetics;
metabolism
- From:
Chinese Journal of Virology
2014;30(3):278-284
- CountryChina
- Language:Chinese
-
Abstract:
In this study, pEGFP-N1 was chosen as the reporter plasmid and transferred into Ctenopharyngodon idellus kidney (CIK) cells by electroporation, and the optimal electroporation conditions were determined by testing the transfection efficiency with different voltages, pulse times, plasmid amounts, and numbers of shocks. The results showed that the maximum electroporation efficiency was achieved under the following conditions in a 0.2 cm electroporation cuvette containing CIK cells (1.5 x 10(7)/mL, 200 microl): electric voltage 200 V, pulse time 45 ms, plasmid 30 microg, and one electric shock. The total genomic RNA of grass carp reovirus (GCRV) was extracted in this experiment and reversely transcribed into cDNA, which was used to amplify the gene segment of GCRV non-structural protein NS26 using designed specific primers. The PCR product was recombined into pEGFP-N1 vector. The fusion protein EGFP-NS26 was successfully and efficiently expressed in the CIK cells by electroporation, which was confirmed by both fluorescent imaging and Western blot analysis. This experiment laid a foundation for further functional studies of the non-structural protein NS26 of GCRV.
