Prokaryotic gene expression and detection of white spot syndrome virus (WSSV)VP28

Abstract:

VP28 is the envelope protein of white spot syndrome virus (WSSV).After the codon optimization synthesis VP28 gene sequence,and enzyme digestion by restriction enzymes Nde I, Xho I, inserted the correct reading frame sequence into the expression vector-pET-28a(+).After the recombinant plasmid into E. coli BL21 (DE3) was digested by double enzyme and sequencing, it was confirmed that we have successfully constructed VP28 genetic prokaryotic expression vector-pET-28a-VP28.The transferred strain induced by IPTG, SDS-PAGE showed that contains the expected size of about 30 ku protein band, which was mainly expressed in inclusion body form. The recombinant protein was purified by Ni-IDA-Sepharose CL-6B affinity chromatography column and we got the purity of 95% protein finally.Gradiently dilute the purified protein as the quantitative standard curve of Western blotting, to quantify the amount of VP28 expression in transgenic Anabaena sp. PCC 7120.The experiment results showed that the recombinant protein and VP28 expressed in Anabaena sp. PCC 7120 had the same molecular weight.Use the purification of recombinant protein as a quantitative western blotting standard curve, calculate the expression of VP28 in transgenic Anabaena sp. PCC 7120.The largest amount was 5.14 μg/mL, on 17th day, accounting for 1.45% of the total protein concentration.This is not only of great significance to efficient cultivation of transgenic cyanobacteria, but also a foundation to determine the effective dosage of oral vaccine for prawn to prevent and cure white spot syndrome in the future.