伪狂犬病毒gD蛋白在水稻细胞中的表达及其纯化条件优化OA

To explore the feasibility of expressing the pseudorabies virus gD protein in rice cells and to lay a founda-tion for developing a pseudorabies virus subunit vaccine,this study optimized and artificially synthesized the pseudor-abies virus gD gene based on rice codon usage bias,constructing the recombinant plant expression vector pCAM-BIA1300-gD.This vector was introduced into rice callus via Agrobacterium-mediated transformation,and resistant calluses were obtained after co-cultivation and dark culture screening.Callus genomic DNA was extracted using the CTAB method,and positive calluses were identified by PCR.Positive calluses were expanded in culture,ground,and added to liquid medium for shake-flask cultivation.Western blot was used to detect the expression of the gD pro-tein.Based on the physicochemical properties of the target protein,purification conditions including pH value,cation exchange resin,and hydrophobic resin were screened and optimized.The results showed that 110 calluses were ob-tained after hygromycin screening,with 92 identified as positive by PCR,yielding a positive rate of 83.6%.Western blot analysis of 35 cell lines revealed successful gD protein expression in 15 lines.Purification via two-step chroma-tography using cation exchange resin 650F and hydrophobic resin GE Butyl 4FF yielded gD protein of certain purity.This study established a foundation for using rice as a bioreactor to produce pseudorabies virus subunit vaccines.