Recombinant Expression Of Three Impotrant White Spot Syndrome Virus (wssv) Envelope Proteins And Their Ability On Disease Control

Since white spot syndrome disease caused by WSSV first broke out in EastAsia, it has been quickly spread across the globe. It became the most severeepidemic virosis to shrimp culture industry worldwide, resulting in tremendouseconomic losses. Still no effective WSSV-controlling approach is available. Virusenvelope proteins play important roles in interacting with host cells, initiating virusinfection or mediating virus intrusion. And it has been proved that some WSSVenvelope proteins are effective in protecting hosts against WSSV when deliveredinto shrimp. This stragety has become a hot research field. In this study,WSSV-controlling strategy with live bacteria as an envelope protein deliverer wasexplored. In the mean time, a profile of functions of two recombinant WSSVenvelope proteins VP281and VP31was preliminarily judged. The followings are themain conclusions.1. By using live Escherichia coli strain DH5α as a deliverer and a prokaryoticconstitutive secretory expression plasmid pBTA1as the expression vector, liverecombinant bacteria that could make VP28or VP28transmembrane-truncatedregion directly interact with the shrimp were constructed and respectively named asDhpV and DhpVB. Expression analysis showed that DhpV and DhpVB couldconstitutively express and secrete the target proteins into the culture supernatant.2. In order to test the protection potential, the live recombinant bacteria DhpVBwere injected into the shrimp Exopalamon carincauda Holthuis for three times andchallenge was performed twice simultaneously at the last two injections of the recombinant bacteria DhpVB. DH5α with empty vector pBTA1was used as controls.No protection differences were observed between recombinant bacteria DhpVB andbacteria without VP28after the first challenge. DhpVB displayed statisticallysignificant advantage over bacteria without VP28after the second challenge withan average RI (Resistance index) of0.67±0.08, compared to0.41±0.09ofbacteria without VP28(P﹤0.05).3. WSSV envelope proteins VP28,VP281and VP31were obtained in the formof inclusion body by using Escherichia coli strain BL21(DE3) pLysS as the hostbacteria and the induced expression plasmid pET-30a(+) as the expression vector.After purification with Ni2+-chelating Sepharose column, the target proteins wererenatured through gradient dialysis. This laid the foundation for exploring VP281and VP31as candidate proteins with protection potential against WSSV.4. By using a prokaryotic constitutive secretory expression plasmid as theexpression vector, the recombinant Escherichia coli strain DH5α that couldconstitutively secrete WSSV envelope protein VP28, VP281or VP31were tried tobe constructed. Recombinant DH5α that could constitutively secrete rVP28andrVP281were constructed successfully and were respectively named DhpVP28andDhpVP281. DhpVP281grew much slowly compared with DhpVP28and it wasguessed that the expression of rVP281probably inhibited the growth of E. coli.However, recombinant bacteria constitutively expressing VP31can not be obtained.In order to find out why recombinant bacteria that constitutively secrete rVP31cannot be constructed, recombinant bacteria BL21(DE3) pLysS were constructed withpET-30a(+) as the expression vector and rVP31was obtained in the form ofinclusion body. After renaturation, the antibacterial activity of rVP31was tested.The result showed that rVP31had the antibacterial activity against Micrococcuslysodeikticus.Live bacteria benefit the sustaining of envelope proteins and secretoryexpression vector can make envelope proteins contact with shrimp directly. Besides, E. coli with a clear genetic background can be used as stimulants, boosting theprotection. Live recombinant bacteria carrying VP28could enhance the shrimpsurvival against WSSV on condition of enough contact between VP28and shrimp.This study provided a new basis for controlling WSSV. Also in this study,theanti-bacteria effect of two recombinant WSSV envelop proteins VP281VP31wereobserved for the first time. These observations may increase the virology knowledge ofWSSV.