Analysis Of Receptor For White Sport Syndrome Virus Using Monoclonal Antibody Against Hemocyte Of Shrimp

By the methods of white spot syndrome virus (WSSV) binding to haemocyte membrane and primary cell culturing, monoclonal antibodies (Mabs) blocking the attachment of WSSV to its receptors are found. Receptor for WSSV is identified by western blot and affinity chromatography. The results is found as following:Mice of Balb/c strain are immunized with haemocytes of Fenneropenaeus chinensis. Immunized mice spleen lymphocytes are fused with P3-X63-Ag8Ul (P3U1) myeloma cells and hybridomas are selected in hypoxanthine-aminopterin-thymidine medium. Mabs are divided into three groups: Mabs in group 1 can react with haemocyte membrane, for example Mab 4D8; Mabs in group 2 wells can react with granules in haemocyte cytoplasm, for example Mabs 5F9; Mabs in group 3 wells can react with both haemocyte membrane and granules in haemocyte cytoplasm, for example Mab 7H12.By DOT BLOT, Mab 4D8 is screened to be able to block the attachment of WSSV-DIG to receptors on the haemocyte membrane. The signal of control 1 (Haemocyte membrane is firstly reacted with culture fluid of P3U1 myeloma cells, then reacts with WSSV-DIG) is the most intensive, control 2 (Haemocyte membrane is firstly combined with Mab 4D8, then reacts with heat-treated WSSV-DIG) hardly has any color. Blocking effects of Mab 4D8 are obviously visible with weaker signals than control 1 and stronger than control 2.Blocking effects of Mab 4D8 is quantificationally identified by enzyme linked immunosorbent assay. Haemocyte membranes first are combined with Mab 4D8 and reacted with WSSV-DIG, OD is measured as 0.256. Culture fluid of P3U1 myeloma cells takes place of Mab 4D8 as control 1, OD is measured as 0.423. Heat-treated WSSV-DIG takes place of WSSV-DIG as control 2, OD is measured as 0.02. The binding ability of WSSV-DIG to haemocyte membranes decreases about 41% due to Mab 4D8 blocking effects. The results indicate that Mab 4D8 is able to block theattachment of WSSV-DIG to some of the receptors on the haemocyte membrane.The experiment of WSSV infecting primary cultured haemocytes is in vitro developed and Mab 4D8 effects of protecting primary cultured haemocytes from infection are confirmed. Cultured haemocytes, which are protected by Mab 4D8, are inoculated with WSSV filtrate and observed under the inverted phase-contrast microscope in the following days. During the first four days of infection, cultured haemocytes protected by Mab 4D8 are in a relatively better condition with no serious CPE than positive control (Haemocytes are firstly incubated by culture fluid of P3U1 myeloma cells and then inculated with WSSV filtrate), which has begun to display haemocytes assembling on the third day. From the 5th day of infection, cultured haemocytes protected by Mab 4D8 begin to display CPE, and positive control has shown serious CPE.Receptor for WSSV blocked by Mab 4D8 is identified by western blot as 175 kDa on the haemocyte of Fenneropenaeus chinensis. On the haemocyte of Marsupenaeus japonicus, Mab 4D8 can recognize a 175 kDa protein just like Fenneropenaeus chinensis, on the haemocyte of Litopenaeus vannamei and Procambarus clarkii, Mab 4D8 can recognize a 200 kDa protein.Mabs against haemocytes of Litopenaeus vannamei are produced by immunizing mice of Balb/c strain with haemocytes of Litopenaeus vannamei. Mabs against haemocytes of Litopenaeus vannamei are divided into 2 group by recognizing types of haemocytes: Mabs in group 1 can recognize three types of haemocytes, for example Mab 1B4. In three haemocyte types, plasma of haemocytes all show visible brown color due to reacting with Mab 1B4 by immunocytochemical method, nuclears all show blue stained with hematoxylin: hyaline haemocytes without granules but with a high nuclear-to-plasma ratio, semi-granular haemocytes with a few granules, granular haemocytes with a number of large granules and a low nuclear-to-plasma ratio. Mabs in group 2 can only recognize granular haemocytes, for example Mab 1H11. In granular haemocytes, plasma of haemocytes show visible brown color due to reacting with Mab 1H11 by immunocytochemical method, nuclears show blue stained with hematoxylin. In hyaline haemocytes and semi-granular haemocytes,plasma of haemocytes show no color and nuclears show blue stained with hematoxylin.Following Mab 1H11 is reacted with haemocytes in WSSV-infected shrimp by immunocytochemical method, the number of total haemocytes and granular haemocytes in infecting group is counted in each visual field of microscope. On the first three days of infection, the number of total haemocytes increase, 150 cells in average of per visual field on the first day, 160 cells on the second day, 190 cells on the third day; the number of granular haemocytes also increase, 22 cells in average of per visual field on the first day, 27 cells on the second day, 87 cells on the third day. Granular haemocytes proportions in total haemocytes also increase, 15.2% on the first day, 18% on the second day, 45.9% on the third day. In the following days, the number of total haemocytes and granular haemocytes both decrease sharply in infecting group, granular haemocytes proportion in total haemocytes decrease from the third day 45.9% to the eighth day 43.3%, but still higher than control group which showes no great difference from start to the end with 15.2%.The first positive reactions with Mab 1B4 against three types of haemocytes are detected in the early stage of embryos. The haemocytes are few in number, mostly isolated. Their morphological appearance is similar to that of haemocytes from adult shrimp. Using Mab 1H11 against granular hameocytes, no labeled cell in samples before zoea HI stage is observed by immunofluorescence staining. For zoea III stage, labeled cells are detected with Mab 1H11 and the numbers of grannular haemocytes are few, but the signal is obvious. At the stage of postlarvae, positive haemocytes with Mab 1H11 are more and the positive intensity is stronger.In conclusion, Mab 4D8 is screened to block the receptor for WSSV, and the receptor is identified as 175kDa, which can help to clarify the mechanism of WSSV. In WSSV-infected shrimp, haemocytes variations are studied and haemocytes ontogenesis are analyzed.