Cloning And Expression Of Envelope Proteins Genes Of Shrimp White Spot Syndrome Virus And Study On The Resistance Of Expression Products In Crayfish

This research is based on WSSV-infected penaeid shrimp, P.chinensis. Wild WSSV was isolated and purified from WSSV-infected tissues of penaeid shrimp and named WSSV-ZJ. The main envelope protein VP28 and VP19 genes were amplified by polymerase chain reaction (PCR) based on virus genome. Then these genes were both expressed in E. coli and in baculovirus-silkworm expression system. In addition, an initial research was made to evaluate the effect of the expression product on white spot syndrome. The result is shown below.1, a batch of WSSV-infected penaeid shrimp was collected from an aquaculture farm located in Xiangshan county (Ningbo, Zhejiang province) in 2001. Crayfish, Cambarus proclarkii, were infected with WSSV-infected shrimp and they almost all died three to ten days post-infection. Using sucrose gradient centrifugation baculovirus were isolated from infected shrimp. Purified virions were observed by electron microscopy and found that the size and morphology is much like white spot syndrome virus. The size of complete virion is about 100X400nm. Based on purified virus, we got the special bands to WSSV after amplifying by PCR, and it is proved that the virus we isolated is WSSV in molecular level.2, VP28 and VP19 genes were obtained by amplification using PCR. The sizes of VP28 and VP19 were 630bp and 380bp. Nucleotide sequence analysis shows that VP28 and VP19 is about 615bp and 366bp in size and they encode postulated proteins of 204 and 121 amino acids, respectively. The VP28 and VP19 gene sequences were analyzed with DNATools software and the result revealed that they are both show a high degree of homology by 99% in nucleotides compared with VP28 and VP19 sequences available in GenBank. But when they are compared with other viral or cellular genes in GenBank, it shows very low relevant homologies.3, VP28 and VP19 genes were expressed in E.coli prokaryotic expression system.Cloned VP28 and VP19 genes were inserted into the multiple cloning site of pET30a vector. The resulting plasmids were named pET30a-vp28 and pET30a-vp19 respectively. PCR and restriction endonuclease analysis proved that these two foreign genes have been inserted into the pET30a vector downstream of its strong promoter lac-Z. After inducing by isopropyl thio- P -D-galactoside (IPTG) at 37C for 4 hours, VP28 gene expression product shows prominent special bands in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the size of expression product is corresponded to the estimated size. It is suggested that VP28 gene of WSSV has been expressed in the E.coli. But VP19 gene expression product shows no special bands in SDS-PAGE and Western blotting.4, In order to gain VP28 and VP19 with the similar structure and biological activity as nature ones, this research employ a baculovirus-silkworm expression system using its powerful promoter (polyhedrin promoter) to highly express the foreign genes. Cloned VP28 and VP19 genes were inserted into the multiple cloning site of pBlueBacHisC vector. The produced plasmids named pBlueBacHisC-vp28 and pBlueBacHisC-vp!9 respectively. PCR and restriction endonuclease analysis shows that these two foreign genes have been inserted into pBlueBacHisC. The recombinant transfer vectors pBlueBacHisC-vp28 and pBlueBacHisC-vp!9 were cotransfected with a new recombinant virus HyNPV (hybrid of AcNPV and BmNPV) which has advantages of BmNPV and AcNPV into Sf-9 insect cells respectively. After homologous recombination, purified recombinant virus HyNPV-vp28 and HyNPV-vpl9 were obtain by selecting white colonies and blue colonies. The recombinant virus can cause insect cell cytopathic effect. The recombinant virus was injected into the cavity of fifth-instar silkworm larvae. During three to four days post-infection the larvae appeared obvious pathological syndrome. By the 5th day, the hemolymph presented yellow and the larvae began to die. Apparent special bands were observed in both SDS-PAGE and Wester...