The Immune Effect Of WSSV-VP28Fusion Protein And Cloning Of F₀-ATP Synthase CDNA

White spot syndrome virus (WSSV) causes huge harm to the shrimp farmingindustry. So far, there have been no effective treatment measures to control WSSVinfection. In order to explore effective prevention and control methods to WSSV,three kinds of fusion protein were constructed and the protective effect of these fusionproteins to L.vannamei was studied. Also, the full length of the sequence of F0ATPsynthase which is related in the process of WSSV infection was cloned in the lastsection. The research will provide the basis for subsequent experiments.1. Primers were designed according to the sequence of vp28C and Hsp70. TheC-terminal Hsp70of peptide-binding domain of Litopenaeus vannamei and white spotsyndrome virus VP28were cloned and ligated to the vector pBAD/gIIIA to constitutefusion gene. The fusion protein was expressed in E.coli Top10cells after inducingwith L-arabinose. The fusion protein was confirmed by SDS-PAGE and Western-blotassay. The higher purity of fusion protein was acquired by Co2+affinitychromatography combined with ultrafiltration technology. The purified fusion proteinwas injected into the Litopenaeus vannamei. Shrimps were infected withWSSV-infected shrimp. The results showed that VP28-Hsp70fusion protein hadprotection effect of L.vannamei against WSSV infection. The transcriptionalexpression changes of related gene were assayed by real-time PCR. The resultsshowed that the transcriptional expression of SOD、LZM、CAT and STAT in thegroup injected with fusion protein is significantly enhanced compared with that of thecontrol group. Administration of VP28-Hsp70p fusion protein could delay thecumulative rate of Litopenaeus vannamei from white spot disease.2. Primers were designed according to the sequence of ferritin gene inLitopenaeus vannamei. Ferritin was attained by PCR amplification. The recombinantvector was successfully constrcuted through double enzyme and connection reactionand transformed to compenent cells Top10. The recombinant plasmid was confirmedby sequencing. The fusion protein was expressed in E.coli Top10cells after a largecultivation and induction with a certain concentration of L-arabinose. The fusionprotein was confirmed by SDS-PAGE and Westerb-blot assay. The higher purity offusion protein was acquired by Co2+affinity chromatography combined withultrafiltration technology. The purified fusion protein was injected into theLitopenaeus vannamei and shrimp were infected with WSSV-infected shrimp afterimmunation with a period of time. The results showed that Fe-VP28C fusion proteinhad better protection effect to Litopenaeus vannamei in the early period ofWSSV-infection. The transcriptional expression changes of LGBP and LvP38wereassayed. The results showed that the transcriptional expression of LGBP and LvP38 in the group injected with fusion protein is significantly enhanced compared with thatof the control group, which further verified the protection effect to the shrimps.3. The cell penetrating peptides (TAT) gene sequence was obtained by theliterature data. Through the online design optimization software, the sequence wasoptimized to make it more suitable for expression in E. coli and synthesized. Therecombinant TAT-VP28C was acquired through double enzyme digestion and ligationreaction. After recombinant plasmid was transformed to E.coli Top10cells, therecombinant protein was expressed successfully by cultivation of a large number ofE.coli and induction with L-arabinose. The rcombinant protein was purified by Co2+affinity chromatography technology. But a lot of protein was precipited in the processof protein renaturation. Subsequent renaturation conditions need further exploration.4. According to the announced partly F0-ATP synthase sequence, the specificprimers was designed. The full length gene of F0-ATP synthase of Litopenaeusvannamei was cloned by rapid amplification of cDNA ends (RACE) method. The fulllength cDNA of F0-ATP synthase had an open reading frame (ORF) of744bpencoding248amino acids and the predicted molecular weight of the mature peptidewas28.2kD. The homology analysis of F0-ATP synthase with other species showedhigher similarity with Caligus clemensi (50%) and Drosophila melanogaster (60%). Inorder to research the distribution of F0-ATP synthase in hemocytes of L.vannamei,flow cytometry analysis was performed. The results showed that F0-ATP synthasewas widely distributed in the surface of hemocytes. These results laid a basis forfurther studies on the role of F0-ATP synthase．...