| White Spot Syndrome Virus (WSSV) is the causative viral pathogen of a deadly disease of cultured shrimp. Four viral attachment proteins of WSSV and the 53kDa shrimp membrane protein which acted with VP37 was identified, which laid a foundation for deeper explanation of pathogenic moleucular mechanism. Binding assay was observed between DIG labeled WSSV and several kinds of primary prawn's cells (lymphoid organ tissue,hemocyte,hematopoietic tissue,heart organ and so on), which proved the fact that the receptors of WSSV existed. And four virus attachment proteins (VAP) of WSSV were identified by overlay shrimp membrane protein (gill and hemocyte) on the WSSV structural polypeptide separated by SDS-PAGE, they were VP 37, VP 39, and two polypeptides which molecular weight was higher than 97 kDa. Mass spectrometric analysis identified VP37 was the product of wsv 254. Then the gene sequence was rebuild according to the tropsim of Pichia pastoris. And the actived recombinant VP37 was abtained by Pichia pastoris express system. The cellular protein against VP37 was found by Ni2+ affinity chromatograph, which was 53 kDa shrimp gill membrane protein. At the same time, WSSV was treated with 2% Tween-20, then purified by CM-Sepharose FF ion exchange Chromatography and Sephadex G-75 gel filtration separately, among the collected fraction, the 53 kDa protein had the binding activity by ELISA test which be consistent with the result above. The sequence of 53kDa highly matched with synthase beta subunit (Drosophila melanogaster) by mass spectrometry analysis. VP 37 was collected by elution, and its antibody was prepared by immunize rabbit. The antibody could react with VP37 specially, and no cross activity with the higher molecular weight proteins of WSSV. The peptide (L I Q E R G D E I) with "R G D" motif of VP37 was synthesized to verify its activity. Knowing that "R G D" was playing an important role in VP37's binding activity, but not standing for all. By treating WSSV with detergent, the purified VP 28 was obtained, which is one of the major structural proteins in WSSV envelope. And VP28 had no binding activity with gill membrane protein of shrimp. Four kinds of tissue (gill, hemocyte, hepatopatopncreas and muscle) of Litopenaeus vannamei were chosen for the extraction of cell membrane protein. This study mainly focuses on the way to obtain membrane proteins and the composition analysis of their polypeptides. And their characteristic polypeptide was 75.0 kDa, 70.5 kDa, 26.7 kDa and 71.2 kDa respectively. The 43 kDa protein always appeared in the SDS-PAGE of purified WSSV. However its identity was actin from host by mass spectrometric analysis. This paper provided theoretical bases on further researches about the 53 kDa gene variation among different species with different WSSV susceptibility. The work laid a foudation for the molecular virological research of WSSV. |
