Molecular characterization of prohibitins and their differential responses to WSSV infection in hemocyte subpopulations of Fenneropenaeus chinensis.

In our previous work, prohibitin1 (PHB1) was identified to be only expressed in granulocytes of Fenneropenaeus chinensis. In order to elucidate the potential immunological properties of prohibitins in hemocyte subpopulations, in this paper, the full-length cDNAs of PHB1 and PHB2 were firstly cloned from F. chinensis using rapid amplification of cDNA ends approach, and they were designated Fc PHB1 and Fc PHB2, respectively. Based on the sequence analysis and multiple sequence alignment, Fc PHB1 and Fc PHB2 were members of SPFH protein family. By quantitative real-time RT-PCR, the higher mRNA transcription levels of Fc PHB1 and Fc PHB2 were detected in intestine and hemocytes of F. chinensis , and these two genes in hemocytes were significantly up-regulated upon WSSV infection. The Fc PHB1 and Fc PHB2 were recombinantly expressed in Escherichia coli BL21 (DE3), and employed as immunogens to produce the polyclonal antibodies (PAbs) in rabbits. Indirect immunofluorescence assay (IFA) revealed that the Fc PHB1 and Fc PHB2 were located both in the cytoplasm and nuclei of hemocytes, which could also be specifically recognized by the PAbs against Fc PHB1 or Fc PHB2 in Western blot. Interestingly, it was found that Fc PHB1 and Fc PHB2 were only expressed in the granulocytes of heathy shrimp and highly expressed in the WSSV-infected granulocytes, however only weak expressions of Fc PHB1 and Fc PHB2 were observed in the hyalinocytes of WSSV-infected shrimp. Meanwhile, silencing of Fc PHB1 and Fc PHB2 genes were performed by small interfering RNA, and the results showed that the WSSV copies in hemocytes were increased by knockdown of either Fc PHB1 or Fc PHB2, and the cumulative mortalities of shrimp in the silenced groups were also markedly increased. These results demonstrated that Fc PHB1 and Fc PHB2 played important roles in anti-WSSV infection, and their differential expression characteristics in hemocyte subpopulations provided a further understanding of the immune functions of granulocytes and hyalinocytes. • The cDNA sequences of PHB1 and PHB2 of Fenneropenaeus chinensis were cloned and characterized. • Fc PHB1 and Fc PHB2 were significantly up-regulated post WSSV infection. • Fc PHB1 and Fc PHB2 play important roles in inhibiting WSSV infection. • Fc PHB1 and Fc PHB2 were differentially expressed in granulocytes and hyalinocytes. [ABSTRACT FROM AUTHOR]