Studying On Function Of Relavant Factor To Immunity Of Procambarus Clarkii And Comparison Of Variable Region Of White Spot Shrimp Virus Isolates

Hemolymph of crayfish(Procambarus clarkii) were collected from the ventral sinuse of crayfish while they were injected white spot shrimp virus(WSSV)at 2h,4h,8h,16h and 32h .Then phenoloxidase(PO) activities were measured by spectrophotometer and properties of hemagglutinins were measured by cross adsorption tests and haemagglutination assays.The results were that PO activities showed an upregulation until 8h p.i.,while at 16 and 32h p.i.the PO activities reduced,but the agglutinating titers that agglutinating activities against chicken erythrocyte had no obvious changes.The results showed while WSSV were multiplicated in the crayfish,it would effect on the PO activities but not on the agglutinating activities.The specific primers were designed referring to the reported gene sequences of GenBank accession number CD644775. Serine proteinase inhibitor gene(PCI188) was amplified by reverse transcription polymerase chain reaction (RT-PCR) method using total RNA isolated from crayfish (Procambarus clarkii )hemolymph as template. The amplified cDNA fragments were purified and inserted into pET-32a vector ,then transformed into E.coli BL21 (DE3)and Rosetta.They induced by IPTG for the expression of recombinant protein. The protein was only expressed in Rosetta other than in BL21.Expressed fusion protein was identified by SDS-PAGE and Western-blotting. The results showed that 50 kD fusion protein was identical with the known protein coded by gene PCI 188 of crayfish.Fusion protein of serine proteinase inhibitor was purified by His Bind Resin purification Kit and used to immunized to rabbit. Then phenoloxidase(PO) activities were measured by spectrophotometer after immunized serum and haemolymph of crayfish were cultivated.The results showed that immunized serum increased the PO activity of crayfish . This experiment is firstly to proved that the protein coded by gene PCI 188 of crayfish can inhibit serine proteinase.The specific primers were designed referring to the reported gene sequences of GenBank of white spot shrimp virus(WSSV)isolates from Thailand,China,Taiwan.Variable region of WSSV isolates from ZHSH,ZHJ,HN,QD1,QD2 was amplified by polymerase chain reaction (PCR) method and aloned,sequenced them.Sequence comparisons and phylogenetic analyses were performed by DNAstar software.We analysed the characteristic of variable regions and then discovered that variable region is closely related to evolution of WSSV.