Cloning Of Envelope Protein VP28 Gene Of WSSV And Lts Expression In Cyanobacteria

WSSV envelope protein VP28 gene was cloned and expressed in cyanobacteria is to identify the mechanism of WSSV entry into the shrimp and prophylaxis and treatment of WSSV in shrimp. White spot syndrome virus (WSSV) is a major virus that is highly virulent in penaeid shrimp; and can also infect most important species of crustacean. WSSV envelope protein VP28, 28kD.Recently, it had been proved to be likely to play a key role in the initial steps of the systemic WSSV. Protein VP28 was chosen to be expressed as antigen in cyanobacteria. This study was inspired by an interesting phenomenon that antivirus compound or viral resistance mechanism may exist in transgenic cyanobacteria. So in this study, we constructed the transgenic cyanobacteria expressing VP28 protein to identify the mechanism of WSSV infection of shrimp and find a new antivirus method with transgenic cyanobacteria.In this study, a stain of WSSV was isolated and purified from P. chinensis with sucrose gradient centrifugation. Envelope protein VP28 was identified by SDS-PAGE. Genome DNA of WSSV was extracted. The primer was designed according to the reported sequence in the GenBank and synthesized. The gene of VP28 protein was cloned from Genome DNA with PCR amplification. The product of PCR digested by restriction enzymes and was cloned in pUC-19. The cloning vector named as pUC-VP28. The gene was sequenced and the results showed that the vp28 gene contains 615 nucleotides encoding 204 amino acids and 100% identity with the vp28 gene sequences reported in GenBank. Then, the vp28 gene inserted at downstream of the promoter PpsbA the recombinant shuttle expression vector for cyanobacteria was named as pRL-VP28. PpsbA is a strong promoter in cyanobecteria to transcript the gene; and the SD sequence added before ATG of vp28 gene have a higher translation lever in cyanobacteria.Then expression vector pRL-VP28 was introduced by triparental conjugative transfer into Anabaena sp. PCC 7120, fresh filament cyanobacteria, Anabaena sp. PCC 7120 in 50% seawater (V/V), a species induced by seawater, and Synechococcus sp. PCC 7002, and marine unicellular cyanobacteria. Transgenic cyanobacteria were obtained under the pressure of antibiotics. In the these three transgenic cyanobacteria, pRL-VP28 can exist as a complete plasmid in Anabaena sp. PCC 7120 and Anabaena sp. PCC 7120 in 50% seawater(V/V),but can only integrate into the chromosome of Synechococcus sp. PCC 7002. Then the transformation phenomenon was compared Anabaena sp. PCC 7120 in 50% seawater with in BG-11. And the morphology and growth of the transgenic cyanobacteria was also observed and measured.Western blotting proved that VP28 protein has been expressed in both transgenic Anabaena sp. PCC 7120, molecular weight is 28 kD.Except for the VP28 gene sequence, no similar reports have been in this field. In our work, we hope the transgenic cyanobacteria can understand the molecular mechanism of the infection of Penaeus Chinensis by WSSV. And the same time, we want to do some basic work on the possibility of the antagonistic mechanism existing cyanobacteria induced by viral key gene and preparation of vaccine to protect host against the virus infection applying cyanobacteria transgenic.