Expression And Making Antiserum Of SARS Coronavirus Gene PU5 And Screening Recombinant Baculovirus Of N,M,E Gene

The complementary DNA of putative uncharacterized gene 5 (PU5) was prepared from SARS coronavirus (SARS-CoV) gene RNA by Revers-Transcription Polymerase Chain Reaction (RT-PCR). The amplified fragment was cloned into the plasmid vector pGEX-6P-1. The recombinant vector was designated pGEX-PU5. The PU5 of SARS-CoV was expressed in E. coli as fusion protein with GST protein at N-terminus. SDS-PAGE result showed that the PU5 gene was expressed in the prokaryotic expressing system, and the target protein take the proportion of 36.5% in the whole protein by scanning assay. The soluble parts of the cell extract were purified by Glutathione SwpharoseTM 4B Kit ,and the purified protein was designated GST-PUP5. GST-PUP5 was cleaved by PreScission TM Protease and got purified PUP5. Additionally, the Western-blot assay proved the GST-PUP5 and PUP5 having good immunological activity. After being made sure of its immunological activity, the GST-PUP5 and PUP5 was emulsified with complete Freund's adjuvant. In order to produce antiserum , BALB/c female mice were vaccinated. The PUP5 can be used to further search the function of this protein . The anti-PUP5 antiserum lay a foundation for making anti-PUP5 antibody and studying diagnosis reagent of distinguishing SARS-CoV. The complementary DNA of 1269bp nucleocapsid gene(N gene), 666bp membrane gene (M gene)and 231bp envelope gene(E gene)were prepared from SARS gene RNA by RT-PCR. The amplified fragments were cloned into the baculovirus transfer vector pBlueBacHis 2A and sequenced.. The positive recombinant transfer vectors were designated pBlueN,pBlueM,pBlueE. They were sequenced and co-transfected the sf9 cell with the Bac-N-BlueTM DNA by the technique of cationic liposome mediated transfection. The recombinant baculovirus were purified by three times of plaques assay with the chromogenic substracte X-gal in the low temperature melting agarose and analyzed by PCR.We get several stains recombinant baculovirus rpBlueN,rpBlueM,rpBlueE. These recombinant baculovirus are used to express N,M and E protein in HighFive insect cell . It is a good base for research and development of the SARS-CoV gene engineering subunit vaccine.