[Expression of human VIGILIN N terminus and observation of subcellular localization].

OBJECTIVE

To express human VIGILIN N terminus gene in E. coli, prepare the polyclonal antibody and study the subcell localization of human VIGILIN.

METHODS

The N-terminal sequence of human VIGILIN was amplified by PCR and cloned into the polyclonal site of pGEX-4T-2 expression vector. The fusion protein was expressed under induction of IPTG in E. coli BL21. The extracted GST fusion protein was purified by GSTrap-FF affinity chromatography. The polyclonal antibody against human VIGILIN N was prepared by immunizing New Zealand white rabbits using the purified fusion protein as immunogen and analyzed the titer and specificity of the antiserum by ELISA and Western blot. Through immunofluorescence staining, the distribution of VIGILIN in cell was observed.

RESULTS

Expression of the GST fusion protein was induced with 1 mmol/L IPTG at 28 C for 3 hours. The antibody titer was 1:16000. Western blot analysis demonstrated that the polyclonal antibody can recognize VIGILIN specifically. VIGILIN present in both the cytoplasm and the nucleus. Its distribution in the nucleus concentrated on the inner layer of nuclear membrane and the region close prominent area of DAPI staining.

CONCLUSION

The human VIGILIN fusion protein was successfully expressed. The polyclonal antibody against human VIGILIN was generated and was further applied to the study of distribution of VIGILIN in cells. This study will provide a substantial base for further clarification of the quality and function of VIGILIN.