| WSSV is one of the most pathogen that causes high mortality in cultured shrimp, ithas a high mortality rate and wide host range. Research on the pathogenicity of WSSVwill be great effective for the control WSSV infection.The main research and the results are as follows:Part1: VP90p expression and its interaction with WSSV and hemocytes werestudied. According to the vp90genetic sequences on GenBank, the primer was designedand synthesized. About758bp of vp90was attained by PCR amplification and ligatedto the expression vector pBAD/gIIIA to constitute recombinants pBAD/gIIIA-vp90.Recombinants were transformed into E.coli Top10and induced by L-Arabinose at37℃.SDS-PAGE and western-blot assay showed that the protein coressonpding to35kDawas induced. Detection of express product by SDS-PAGE and Western-blot test, showsthe expected objective35kDa protein. Purified VP90p was attained by means of Co2+affinity chromatography. Far-western and matrix-assisted laser desorption ionizationmass spectrometry (MALDI-MS) assay showed that VP90p interacts with VP26andVP28, two major envelope proteins. Far-western blot with recombinant VP26(rVP26),N-terminal end of VP28(rVP28N) and C-terminal end of VP28(rVP28C) providedfurther evidence for interaction of rVP90and rVP26or rVP28. These results suggestthat VP90p is anchored to the envelope through interacting with VP26and VP28.For explore the interaction of VP90p with hemocytes of shrimp, FITC-VP90p wasincubated with hemocytes of L.vannamei, Confluorescence observation showed thatVP90p interact with hemocytes.Part2: Interactions of rVP39with WSSV and gills membrane protein wasinvestigated. Western-blot and matrix-assisted laser desorption ionization (MALDI) MSassays showed that VP39interacted with VP26, a major envelope protein. Far-westernblot provided further evidence for interaction between VP39and VP26. These resultssuggested that VP39anchor to the envelope through interacting with VP26.Furthermore, one protein was selected by incubating FITC-VP39with gills membrane protein of L.vannamei and F0-ATP synthase b lane was identified by means ofMALDI-MS assay.Part3: F0-ATP synthase b lane expression and function assay were studied in thischapter. Primer was designed according to F0-ATP synthase b chain sequence, and230bp gene was got through PCR amplicfaction. Recombinant pBAD/gIIIA-F0wascontrcuted and transformed into Top10. Corrected recombinants were induced with L-Arabinose. SDS-PAGE and western-blot assay showed that the protein coressonpding to14kDa was expressed. Purified VP90p was attained by means of Co2+affinitychromatography and used to produce anti-rabbit antibody.Far-western blot assay showed that F0-ATP synthase b lane could interact withrVP26, rVP28C, rVP28N and rVP39. Cellular localization of F0-ATP synthase b laneand VP26, VP28, VP39by indirect immunofluorescence assay indicated that they allco-located on the surface of the hemocytes. The relative protection rate of F0-ATPsynthase b lane to infected shrimp is17.94%. Tissue distribution analysis indicated thatF0-ATP synthase b lane is widely distributed in muscle, hepatopancreas, intestine,blood lymph, lymphoid and gill tissues. Cellular localization analysis revealed thatF0-ATP synthase b lane distributed on blood lymphocyte plasma membrane. Real-timePCR analysis showed that the mRNA levels of F0-ATP synthase b lane increasedrapidly and reached the highest in tissues of intestines, gills, hepatopancreas andhemolymph after infection for12hours.Part4: QDs-Labelled VP37p interact with Primary Hemocyte Culture ofFenneropenaeus chinensis.Culturing of primary hemocyte from Fenneropenaeuschinensis was reported. Interaction between QDs-labeled binding protein VP37p ofwhite spot syndrome virus and primary hemocyte was observed. Results showed thatprimary hemocyte culture maintained for12days. QDs-labeled VP37p interacted withprimary hemocyte. |
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