FUSION-EXPRESSION AND BIOACTIVITY ANALYSIS OF CARP GROWTH HORMONE GENE AND ENVELOPE PROTEIN VP28 OF PENAEUS MONODON WSSV

Carp growth hormone is a secreted protein from growth gland for promoting carp growth. White spot syndrome virus (WSSV) VP28 is one of the envelope proteins acting as a key factor to infect the shrimp. Two pairs of primers are designed and synthesized respectively according to the sequence of gene gh and vp28. The gh and vp28 DNA fragmentswere amplified by PCR respectively.Gene gh and vp28 were fused and cloned into yeast shuttle vector pPIC6AC at the site of polyclone resulting to recombinant vector pPIC6AC-(gh+ vp 28), gene gh lying in 5c end and gene vp 28 in 3cend. Shuttle vector pPIC6AC-(gh+ vp28) were lineaized with Bstx1 and transformed into P. pastoris X-33 strain. Incubated transformant on YPD plate with 300mg/mL blasticidin at 30 e for 72h. Selected positive clone and extracted yeast genome for ident ifying with PCR by general primer AOX1, primer 1, 2, 3 and 4, respectively.Results showed that interest gene was introduced into target site of P. pastoris X-33 genome by homologous recombinat ion.After incubated in YPDmedium at 30e for 24h, 4% yeast volume were transferred into BMGY med-ium to incubate. When OD600 reached 4, engineered yeast were induced by methanol at ultimate concentration 0. 5% with one time every 24h for continuous 4 days. Fusion protein (GH+ VP28) is expressed and secreted out from host yeast. Supernatant were collected after centrifugation at 4800 r/min for 10min at 4 e. The molecular weight of the fusion engineered protein was about 66kDa,which was identified by SDS-PAGE and Western Blot analysis. The fusion engineered protein (GH+ VP28) with 6 @ his tag at the C end was purified by Ni2+-column chromatography and determined concentration with Bradford kit. Protein (GH+VP28) was injected into two groups of crayfish to test activity of st imulating growth and protecting against wssv independently. Test data of stimulating growth were collected and analyzed with testing significance of difference of T inspection after 5 weeks, and test data of protection against wssv were instructed by accumulative mortality curve after 19 days. Both of the test data indicated that the protein (GH+ VP28) either stimulated crayfish growth by 20% percent faster than control and difference in statistics between test and control group was significant or protected crayfish against wssv to increase livability by 65% percent. Results showed that the fusion engineered protein (GH + VP28) had the effects of crayfish stimulation growth and protection against wssv. As stimulation-growth agent,we employed method of injecting to test its effect rather than oral in order to take effect as hormone. Though effect as st imulation-growth agent was not distinct, result still was exciting, and we took a valuable step for exploring carp GH to apply on crayfish breed industry. As gene-engineering bioactivity polypept ide, protein (GH+ VP28) protecte crayfish against WSSV acting as subunit vaccine. Protein (GH+ VP28) supplied lower protection with 35% mortality than eng-ineered protein vp 28and vp 28-vp29 with 30%121We analyzed that natural structure of protein (GH+ VP28) was influenced in some degree due to fusion, thereby, its function or bioactivity decreased correspondingly, nonetheless, test elicited us new thoughts to going further for exploring function of fusion engineered protein (GH+ VP28).