| Rabbit hemorrhagic disease (RHD) is a highly contagious and fatal disease characterized by a hemorrhagic syndrome that affects domesticated and wild rabbits of the genus Oryctolagus cuniculus. RHD has a high mortality rate and is responsible for large economic losses to rabbit producers. The disease was first described in China in 1984 and is currently endemic in most parts of the world. Until now, the attempts at in vitro propagation of rabbit hemorrhagic disease virus (RHDV) failed. So at the moment, there are several commercially available vaccines against RHD on the market, but all are elaborated from tissues collected from experimentally infected rabbits. Although they have proved to be effective tools for prevention of the disease, manufacturing this kind of killed vaccines gives rise to many problems resulting from the use of infectious virus and the risk of its dissemination from vaccine factories. Thus, it is important to develop alternative approaches for producing vaccines. To this aim, current research is dedicated to avoid such limitations by expression of VP60, the major structural protein of RHDV using Lactobacillus casei 393 as an antigen delivery vehicle to produce useful vaccine.The rabbit hemorrhagic disease virus was obtained in the liver sample from a dead rabbit. Then, the VP60 gene was amplified by reverse transcript polymerase chain reaction (RT-PCR) and cloned into pMD18-T vector for sequencing. The sequence analysis showed that the nucleotide sequence of VP60 gene of 09-SD was 1740 bp long and encoded a protein of 579 amino acids. The accession number of this sequence to GeneBank is GU564448.Sequence comparison with other published RHDV VP60 genes showed that the homology of the amino acids was between 94.1% and 99.7%.The result showed that the RHDV VP60 gene was highly conserved. The amino acids homology between the RHDV and EBHSV groups was about 75%, but the rate between the RHDV and RCV groups is above 90%.It showed that RHDV and RCV are more closely related to each other than any other calicivirus.In this study, the selective gene VP60 was directionally inserted into the surface expression vector pPG-1 and secretory expression vector pPG-2, constructing recombinant strains pPG-1-VP60/L.casei 393 and pPG-2-VP60/L.casei 393, respectively. The recombinant strain pPG-1-VP60/L.casei 393 of cell surface expression were induced and a protein of about 80KD was detected with SDS-PAGE, which was according with the theoretic molecule weight. Western blot analysis indicated that the expressed protein possessed the antigenic specificity which could be recognized by rabbit anti-RHDV serum. The indirect immunofluorescence test showed that the expressed protein was located on the cell surface of L.casei 393. The recombinant strain pPG-2-VP60/L.casei 393 of secretion expression was induced by 1% lactose in MRS medium and a protein of about 70KD was detected via SDS-PAGE in induced recombinant strain and culture supernatants according with the theoretic molecule weight. Western-blot analysis indicated that the expressed protein possessed the same antigenic specificity as the native virus protein. The experiment indicated that the interest protein was expressed and secreted into the culture supernatants.To identify whether the recombinant strains have the ability to induce systemic and mucosal antibody responses, four groups of non-immunized rabbit were immunized. They were immunized via oral route with pPG-1-VP60/L casei 393, pPG-2-VP60/L.casei 393, L.casei 393 and sterile normal saline respectively. Every rabbit received a dose of 5ml 1×1010 colony-forming units (c.f.u.) of recombinant strains. The immune protocol was administered on three consecutive days biweekly. We collected serum, stool and nasal cavity lavage of the rabbit at different time point after immunization, detected special sIgA level in the nasal cavity lavage and the stool using indirect ELISA, the special anti-VP60 antibody in the serum was detected by haemagglutination inhibition test and indirect ELISA. The HI titer of immunized rabbit can reach to 24-26.The results of indirect ELISA showed that the nasal cavity lavage and stool of the rabbits produce different titers of slgA and that the stool produced higher titer with the highest to be 0.746,while the highest titer of lavage was only 0.658. Statistical software SPSS 16.0 analysis showed that groups immunized with recombinant pPG-1-VP60/L.casei 393 and pPG-2-VP60/L casei 393 showed great difference(p<0.01) with control groups that immunized with L.casei 393 and PBS and that the recombinant secretory strain produced better immunological effect than cell surface expression strain. The results suggested that the recombinant Lactobacillus casei strain could induce systemic humoral immunity as well as effective topical mucosa immune response. And oral immunity administered to rabbits received effective immunity response. After challenge with 1 ml of infectious RHDV, two control groups all died within 72h while all of the immunized ones survived except for a special case.The study indicated that the recombinant strains constructed in this study could induce both mucosal immune and system immune responses. And it can induce the immunity protection in group.
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