Eukaryotic Expression Of Five Genes Of RSV And GFP-CP GFP-SP Fusionprotein In Insect Cells Sf9

In our research, five genes: NS₂, NS₃, CP (Coat protein), SP (Disease-specific protein) and NSvc₄ encoded by RSV (Rice stripe virus) were expressed by the "AcMNPV-sf9 insect cells " eukaryotic expression system (Autographa California nuclear polyhedrosis virus, AcMNPV; Spodoptera frugiperda, sf9) . And the recombinant system was discussed , constructed and optimal to get active RSV protein. The GFP-CP (Green fluorescence protein, GFP; Coat protein of RSV, CP),GFP-SP (Disease special protein of RSV) fusion genes were expressed in sf9 insect cells to observe their expression directly.First, the genes NS₂, NS₃, CP, SP and NSvc₄ of RSV were cloned into pMD18T through RT-PCR (reverse transcription-polymerase chain reaction) approach by inserting the DNA segments in the multiple cloning sites (MCS), named as pMD18T-X (pMD18T-NS₂, pMD18T-NS₃, pMD18T-CP, pMD18T-SP and pMD18T-NSvc₄) . Then the recombinant plasmid pFastBacHTb-X (pFastBacHTb-NS₂, pFastBacHTb-NS₃, pFastBacHTb-CP, pFastBacHTb-SP and pFastBacHTb-NSvc₄) was constructed by double digesting in XbaI/HindIII sites of the transposing vector pFastBacHTb and recombinant plasmid pMD18T-NS₂, pMD18T-NS₃, pMD18T-CP and pMD18T-SP, and double digesting in the KpIII/HindIII sites for pFastBacHTb-NSvc₄．Each plasmid was sequenced to ensure that the target gene is correctly inserted and in right reading frame. After transformation, pFastBacHTb-X was introducted into the competent cells (E.coli DH10Bac) containing a shuttle vector-bacmid to produce recominant baculovirus rb-X (rb-NS₂, rb-NS₃, rb-CP, rb-SP and rb-NSvc₄) . rb-X was isolated and transfected into the sf9 cells to produce the recombinant virus named as P₁-X. An increased diameter, g(?)nular appearance and cells lysis, which were much different from the morphology of normal sf9 celi were observed under fluorescence invert microscope (200×), 24 h～48 h after infection. Fresh insect (sf9 cells were infected with P₁-X containing target genes to amplify viral stocks. And Pn-X was harvested,(?)hich was added at a MOI(multiplicity of infection) of 5 after 4 times of reinfection. There were more than ten visible plaques in a well of 6-well plate to every dilution by the viral plaque assay experiment, which means the title of the five kinds of recombinant baculovirus was more than 1×10⁷pfu/mL (plaque forming units/mL). The culture cells were collected at 48 hpi～96 hpi (hours post-infected) for SDS-PAGE analysis and the result showed five bands with apparent molecular weighs of 28．2 kD, 29．2 kD, 40．2 kD, 25．2 kD and 37．2 kD. There's no band was detected in the suspension by SDS-PAGE. For P₄-CP and P₄-SP, the concentration of special bands were always 5×marker in 48 hpi～96 hpi sf9 insect cells by SDS-PAGE. All these result showed that the five protein encoded by RSV were non-secret protein and they could be expressed through the "AcMNPV-sf9 insect cells" system; During 24 h～72 h, increasing protein would be expressed; CP and SP can express rapidly and steadly by the system. Specificities of NS₂, CP and SP were demonstrated by Western blotting. The protein encoded by NS₃ were purified by Ni⁺-NTA. By indirect ELISA the antiserums against the NS₃ protein were produced in big-ear white rabbits at high titer of 1:9600．The GFP (Green fluorescence protein) and SP, CP genes of Rice stripe virus (RSV) were combinente as a fusion gene with overiap-PCR approach and the fuse gene was inserted into pMD18T at XbaI / HindIII sites to produce pMD18T-GFP-SP and pMD18T-GFP-CP. A XbaI/ HindIII fragment was released from them and placed into a transposing vector pFastBacHTb predigested with the XbaI and HindIII restriction enzymes to produce pFastBacHTb-GFP-X (pFastBacHTb-GFP-SP and pFastBacHTb-GFP-CP). Restriction endonuclease and sequencing analysis verified that the target genes are correctly inserted in right reading frame. pFastBacHTb-GFP-X was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector, bacmid and recombinant bacmid rb-GFP-X (rb-GFP-CP and rb-GFP-SP) was isolated and transfected into the Spodoptera frugiperda (sf9) cells. An increased diameter, granular appearance and cells lysis, which were much different from the morphology of normal sf9 cells, were observed under fluorescence invert microscope 24 h-48 h after infection. And 72 hpi cells showed more green fluorescence could be seen from some cells after 24 h～48 h infected by rb-GFP,rb-GFP-CP and rb-GFP-SP. But the green fluorescence from cells infected by rb-GFP-SP centralized in cytoplasm, which was different from the green fluorescence from cells infected by rb-GFP and rb-GFP-CP. Laser Confocus Microscope observation also showed the same result. And at the same time cell lysis seriously and "inclusion bodies" could be seen from these cells. All these result showed that GFP-X fusion gene was successfully expressed in sf9 cells. And what we do provided some ideas for the study of protein lacation in cells.