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Expression of Some Veterinary Antigens in Silkworm and shRNAs Expression for Enhancing the Resistance of Bombvx Mori to NPV in Vitro and in Vivo

Author: Bhaskar Roy（RuoYi）
Tutor: ZuoYunGen
School: Zhejiang University
Course: Special Economic Animal Feeding
Keywords: Silkworm Bombyx mori (Bm) Bm cell line RNA interference short hairpinRNA Bm nucleopolyhedrovirus (BmNPV) Bm U6promoter Baculovirus expression systemClassical swine fever virus Subunit vaccine
CLC: S884.5
Type: PhD thesis
Year: 2012
Downloads: 22
Quote: 0
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Abstract


Silkworm belongs to the order Lepidoptera, an herbivorous insect species that feeds on mulberry leaves. Because of its economic importance in silk production, the silkworm has been domesticated for thousands of years. Furthermore, in recent years, the silkworm has become an ideal multicellular eukaryotic model system for basic researches, such as genetic, pathogenetic, physiological and molecular biology. Also, the silkworm larvae have a lot of advantages as a biofactory because of its easily reared on large scale at much lower cost, its large and easy to manipulate and relatively short life cycle, and its clear genetic and biological background.Sericulture industry has been suffering huge economic losses every year due to lethal infections by Bombyx mori nucleopolyhedrovirus (BmNPV). The first part of this thesis deals with the inhibition of replication or multiplication of silkworm BmNPV by targeting shRNAs to enhance the resistance of Bombyx mori (Bm) to nucleopolyhedrovirus in vitro and in vivo. We targeted five and four different positions of the genes p143and p35, respectively (p143A,p143B, p143C, p143D, p143E and;p35A, p35B, p35C, p35D). The shRNA presents a7to9nucleotide hairpin loop (5’-TTCAAGAGA-3:) and the19base pair antisense sequence of the target site. The shRNA expression vectors driven by the piggyBac plasmid PXL-BACII, present of BmU6promoter were constructed, and termed was PXL-BACII-EGFP-BmU6-shRNAs. The most effective suppression was observed in the plasmid targeting p143E gene, which suppressed the expression to about90%, and p35C gene which suppressed the expression to about70%in Bm cells. This result suggests that gene suppressing differs on the suitable sequence. Further, we examined the density of BmNPV polyhedra in the infected hemolymph in each larva transfected with both of BmKPV and shRNA plasmids. The results showed that p35C is less polyhedral than other infection of shRNA and also showed that the polyhedra were less than BmNPV infected silkworm. We examined relative expression level of the replicated BmNPV genes in silkworm larvae, hemolymph and BmN cells. Some of the shRNA worked successfully and showed less multiplication of BmNPV. This RNAi system will be useful for suppression of BmNPV in silkworm. It might eradicate BmNPV in silkworm body in the future which will be valuable for silk industr Silkworm is ideal bioreactor for large scale expression of eukaryotic proteins. In most cases, posttranslational processing of eukaryotic proteins expressed in insect cells is similar to protein processing in mammalian cells. Classical swine fever (CSFV) causes significant losses in pig industry in many countries. The E2glycoprotein of CSFV is the main target for inducing neutralizing antibodies and protective immunity in then natural host. We expressed the E2glycoprotein of CSFV in Bombyx mori using the Bac-to-Bac expression system. E2gene was amplified and cloned by RT-PCR and pMD18-T simple vector system. The gene was ligated in the pFastBacHTb vector. We made recombinant DNA into E. coli DH10Bac to produce bacmid. The Bm cell line and silkworm were then transfected or infected with the recombinant rBacmid/BmNPV/E2baculovirus. The SDS-PAGE and western blotting showed an around39.6kDa of band was identified in the hemolymph of silkworm infected with recombinant rBacmid/BmNPV/E2baculovirus, which was consisted to the size of E2glycoprotein. These findings revealed that bacmid expression system can be used as an alternative strategy to develop the drug against CSFV of swine, thereby furthering the mechanistic and therapeutic studies against swine virus.In addition, we also cloned the interferon (INF)-gamma and E2genes from the sus scrofa (Pig) and CSFV genomes, respectively. The INF-gamma and E2genes were linked and sub-cloned into the pFastBacHTb vector and further make the recombinant virus, rBacmid/BmNPV/E2-INFy in E. coli DH10Bac/BmNPV. Silkworm larvae infected with recombinant virus expressed a fused protein of59kDa consisting of E2and INF-gamma subunits. Our results provide evidence that bacmid-based baculovirus expression system could be used as an alternative strategy to develop the subunit vaccine or antigen against CSFV of swine, which will be beneficial to the mechanistic and therapeutic studies against swine virus.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Sericulture > Pest and Disease Control of the silkworm > Virus disease ( pus disease)
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