Role Of Litopenaeus Vannamei Dscam (LvDdscam) In Adaptive Immune Interaction And Phagocytosis

With the study of invertebrate antiviral immune defense mechanism, there has been increasing evidence showing that in addition to the innate immune system, invertebrates also exhibit some kind of vertebrate antigen-antibody-like specific immune responses. To date, however, the data on pathogen-specific invertebrate immune factor is very limited, and the mechanisms by which invertebrates are able to achieve these immune responses are poorly understood.In arthropods, such as fruit fly, mosquitoes, shrimp, daphnia etc., recent reports have pointed to a pathogen-specific receptor with high diversity belonged to the immunoglobulin superfamily (IgSF), the Down syndrome cell adhesion molecule (Dscam). The function of this protein is similar to pattern recognition receptors (PRR). To date, hypervariable Dscam (Dscam-hv) has only been found in arthropod Dscams. By alternative splicing Dscam can generate specific isoforms for different pathogens. Each isoform can only be recognized and bind to homology pathogen-associated molecule, and subsequently elicit the host immune responses. In previous work, we reported that the recombinant of Bacillus subtilis strain harboring VP28with the ability of high-level secretion could evoke specific protection of Litopenaeus vannamei against WSSV by oral delivery. Furthermore, we focus on the immune-related protein Dscam for researching the molecular mechanism of adaptive immunity. We have cloned and analyzed the variable regions of LvDscam after oral delivery of recombinant B.subtilis-VP28.Here, we studyed the interaction between LvDscam specific splice isoforms after oral delivery of rVP28-bs and VP28protein by using yeast two-hybrid system, and further investigated the revelance between LvDscam and specific phagocytosis by RNAi.1、We synthesized five prey genes (AD-Ig2, AD-VP28-Ig2, AD-VIg2, AD-Ig3, AD-VP28-Ig3) obtained from the previous study and performed a yeast two-hybrid to screen these variable domains interacting with VP28to reveal mechanism and biological function of Dscam in response to pathogen in shrimp. The bait gene VP28fragment was fused in-frame with the Gal4DNA-binding domain (BD) of pGBKT7vector and transformed into the Y2HGold yeast cells. While, other prey fragments were fused in the frame with the Ga14activation domain (AD) of pGADT7vector and transformed into the Y187yeast cells. Then the two yeast strains were mating and plated on QDO/X/A.For this assay, all the five independent experiments (VP28+Ig2, VP28+Ig3, VP28+VP28-Ig2, VP28+VP28-Ig3, VP28+vIg2and VP2-8+vIg2) produced blue colonies on SD/-Ade/-His/-Leu/-Trp/X-a-Gal/Ab-A (QDO/X/A) plates. This result indicated that VP28can effect ively interact with the five prey fragments (AD-Ig2, AD-VP28-Ig2, AD-VIg2, AD-Ig3, AD-VP28-Ig3). However, compared to positive c ontrol that Murine p53(pGBKT7-53) interacting with SV40large T-antigen (pGADT7-T), the five groups had fewer colonies and light er color. It may result from the weaker interactions.2、RNA interference was used to silence the Dscam gene of shrimp, so that the transcription of the Dscam gene was inhibited. Then the Real-time PCR was used to detect the effect of interference. In this study two synthesized siRNA sequences as well as PBS buffer as control were injected to Litopenaeus vannamei. The total RNAs of different organs and tissues were extracted after injection for4h,8h,12h,24h,48h,72h and84h, and then we detected the effect of interference by Real-time PCR. The result showed that synthetic siRNA2sequence had better interference effect, while the interference effect of siRNA1was not obvious. After injection of Dscam-siRNA sequence for about12h, the transcription of Dscam gene was inhibited obviously. After24h,48h and72h, mRNA expression levels were maintained in the low level, until84h the effect of transcription inhibition began to eliminate gradually. This was probably due to the instability of siRNA sequence and it could be gradually degraded in the host.3、In order to reveal that Dscam-mediated phagocytosis may play an important role in the adaptive immune system of shrimp, we explored the differences of phagocytic ability induced by WSSV through different immune groups, of which Dscam was silenced by RNAi. The result showed that the percentage of phagocytic of shrimp towards WSSV was regulated by the strength of RNAi. When the transcription of Dscam was inhibited, there was a measurable reduction in the percentage of phagocytic. Meanwhile, the percentage of phagocytic increased when the effect of inhibition was removed. This indicated that LvDscam was involved in specific phagocytosis, and played a significant role in antiviral immunity of Litopenaeus vannamei.In addition, the shrimp which were immuned by B.subtilis-VP28and PBS (control) were both injected with Dscam-siRNA and control-siRNA. We found that the specific phagocytosis towards WSSV was affected by RNA interference of Dscam gene. In conclusion, LvDscam can contribute to the mediation of specific phagocytosis, and thus lead to the selective protection of shrimp after B. subtilis-VP28’vaccination’ against WSSV.