Genetic Prokaryotic Expression And Detection Of White Spot Syndrome Virus(WSSV)

White spot syndrome has become a great disaster for the shrimp industry around the world since 1992 this kind of virus was found in Taiwan and spread to nearly all Asian countries, Europe and America in a short period. O nce the shrimp infects white spot syndrome virus, the mortality will reach 100% from 3 to 10 days, however no efficient protection method was available. Healthful aquiculture environment, selection of seeding and anti- virus poison prevented infection. Since WSSV sequencing project has succeeded, increasing number of researchers studied the virulence mechanisms and demonstrated that virus envelope protein plays the crude role, especially the VP28 protein. VP28 has been expressed in different systems and provided efficient protection against WSSV infection. Recombinant VP28 vaccine has become a promising method for treatment against WSSV. However,VP28 expressed by Escherichia coli must be purified for vaccine preparation and commercial development was limited. However, cyanobacteria has simple structure, fast growth rate and mass recombinant protein production. Thus, cyanobacteria has been engineered as model tool for recombinant protein, bio- fuel and environmental protection. Also, shrimp feed on plant in as young seeding and cyanobacteria is their food supply. Engineered cyanobacteria obtained various advantages for preparing oral administration. Engineered Anabena sp. PCC 7120 habouring vp28 gene has been constructed and drug effect showed anti-WSSV ability. However, expression of VP28 and dose of feed are still not available.Definite technical route was available now. First, vp28 sequence synthesis was optimized by codon. Nde I and Xho I were both inserted as endonuclease sites linking p ET-28 a expressing plasmid. DH5α was transformed and positive clone was sifted. Electrophoresis and double anzyme digestion were prepared for DNA test. Finally, p ET-28a-vp28 expressing plasmid was constructed. Using IPTG to induce expression of inclusion body and was dissolved for further purification as chromatography.95% purity quotient recombinant protein was acquired.Second,amino acid sequence was analyzed for obtaining second structure, trans- membrane domain, signal peptide and antigenicity. Eight epitopes were selected for synthesis polypeptide and coupling K LH and preparing antigen. Regular method immunized mouse for obtaining antiserum 1:16000 BALB/c mouse hybrid tumor cross spleen. Limiting dilution for screening positive clone and three monoclonal stain were acquired: E9, A7 and H3. Ascitic fluid was prepared and subtype Ig G provided valences as 1×10-6、1×10-5、 1×10-4 with epiposition: NGKSDAQMKEED( 84-95AA)、DNIETNMDENLR(42-53AA)、SSFTPVSIDEDE(156-167).Third, proto-expressed p ET-28a-vp28 recombinant protein(0.05μg、0.1μg、0.25μg、0.5μg、1μg、2μg) was set as X axis. Banding signals were captured by Chemi-Doc MP Imaging System and graded by intensity as Y axis. Standard curve was plotted and VP28 expression was calculated. The results showed engineered Anabena showed highest expression rate on the 17 t h day with 5.14 μg/ml as 5% of total protein. However, the highest total protein did not combined with the highest VP28 expression. Three groups of data showed the growth similarity between wide stain and engineered stain. Growth rate of engineered stain was 95.30% o f wide stain. Maximum protein and biomass accumulation did not show in the same growth phase. Thus,VP28 expression rule and optimal culture conditions showed significance for harvesting engineered Anabena sp. PCC 7120.