家蚕蛹表达的重组Vp28疫苗对克氏原螯虾的抗病毒保护效应

对虾白斑综合症病毒(WSSV)的致病性强、危害性大、地域分布和宿主范围广泛,目前还不能有效地控制疫情。将含有WSSV囊膜蛋白Vp28基因的重组杆状病毒HyNPV-Vp28感染家蚕(Bombyx mori)蛹,对发病蚕血淋巴进行SDS-PAGE和Western blotting分析,结果表明Vp28在家蚕体内得到了表达。将重组病毒囊膜蛋白rVp28疫苗配制成药饵,持续口服免疫75天,对克氏原螯虾进行预防WSSV,实验虾分为2%重组Vp28疫苗、2%普通蚕蛹组织匀浆(阳性对照)和普通饵料(阴性对照)3个处理组。免疫35天后进行口服攻毒,20天内rVp28疫苗组的累积存活率为63.33%,与阳性和阴性对照比差异显著(P<0.05),PRP分别达54.16%和59.26%;注射攻毒后20 天内rVp28疫苗组的累积存活率与阳性和阴性对照组比差异不显著(P>0.05),PRP分别为46.12% 和49.99%。第55天对存活虾再口服攻毒,20天内rVp28疫苗组与阳性和阴性对照组比累积存活率差异显著(P<0.05),PRP分别为55.80%和63.16%;二次注射攻毒后,rVp28疫苗组的PRP均为31.25%。对vVp28疫苗组存活虾的胃、肠和肝胰腺组织进行病毒的原位杂交检测均呈阴性反应,而对照组死亡虾组织都呈阳性反应。本研究表明,口服免疫家蚕蛹表达的病毒囊膜蛋白Vp28能诱导螯虾产生抗病毒保护作用,对应用疫苗预防对虾的病毒性疾病具有重要意义。     

家蚕蛹表达的重组Vp28疫苗对克氏原螯虾免疫反应的影响

将含有对虾白斑综合征病毒囊膜蛋白Vp28基因的重组杆状病毒HyNPV-Vp28感染家蚕(Bombyx mori)蛹,配制成药饵持续口服免疫克氏原螯虾35天后,螯虾血细胞的吞噬百分比和吞噬指数比对照组显著提高(P＜0．05);血清中的抗菌活力、溶菌活力、酚氧化酶活性、超氧化物歧化酶活性以及血清和肝胰腺组织中的酸性磷酸酶、碱性磷酸酶活性均显著提高(P＜0．05)。免疫35天后进行口服攻毒,20天内rVp28疫苗组的累积存活率达66．67%,与对照组和对照蚕蛹组比差异显著(P＜0．05),PRP分别达64．29%和58．33%。rVp28疫苗组存活虾的胃、肠、鳃、甲壳下上皮和肝胰腺等病毒侵染的靶组织进行组织病理检测均无病毒感染,DIG标记核酸探针斑点杂交和PCR检测也呈阴性反应;而试验濒死虾的组织都呈现典型的病变特征,病毒DNA检测均为阳性反应。本研究表明,口服免疫家蚕蛹表达的囊膜蛋白Vp28具有增强螯虾机体免疫功能的作用,对应用免疫措施预防对虾的病毒性疾病具有重要意义。     

Effects of oral recombinant VP28 expressed in silkworm (Bombyx mori) pupa on immune response and disease resistance of Procambarus clarkii

The envelope protein VP28 of white spot syndrome virus (WSSV) was overexpressed in the silkworm Bombyx mori, which was achieved by using a baculovirus (HyNPV) expression system and by making silkworm pupa as an alternative host, and then it was directly supplemented in diet at a dose of 20 g kg⁻¹ without purification. During a 30 day feeding period, the levels of phenoloxidase (PO) and superoxide dismutase (SOD) in the haemolymph of the tested Procambarus clarkii increased greatly (P < 0.05) when compared to the control crayfish fed with wild-type HyNPV baculovirus-infected silkworms or normal silkworms. Compared with two controls, the crayfish which had been infected for 20 days showed a significantly lower (P < 0.05) mean cumulative mortality (15.6%), which respectively, resulted in relative percent survivals (RPS) of 83.7 and 84.4%. The efficacy to inhibition of viral infection was further studied by in situ hybridization with a WSSV-specific DNA probe. The high levels of PO and SOD might be important for developing resistance against WSSV in these crayfish.     

Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles

Aims: To achieve high‐level expression and secretion of active VP28 directed by a processing‐efficient signal peptide in Bacillus subtilis WB600 and exploit the possibility of obtaining an oral vaccine against white spot syndrome virus (WSSV) using vegetative cells or spores as delivery vehicles. Methods and Results: The polymerase chain reaction (PCR)‐amplified vp28 gene was inserted into a shuttle expression vector with a novel signal peptide sequence. After electro‐transformation, time‐courses for recombinant VP28 (rVP28) secretion level in B. subtilis WB600 were analysed. Crayfish were divided into three groups subsequently challenged by 7‐h immersion at different time points after vaccination. Subgroups including 20 inter‐moult crayfish with an average weight of 15 g in triplicate were vaccinated by feeding coated food pellets with vegetative cells or spores for 20 days. Vaccination trials showed that rVP28 by spore delivery induced a higher resistance than using vegetative cells. Challenged at 14 days postvaccination, the relative per cent survival (RPS) values of groups of rVP28‐bv and rVP28‐bs was 51·7% and 78·3%, respectively. Conclusions: The recombinant B. subtilis strain with the ability of high‐level secretion of rVP28 can evoke protection of crayfish against WSSV by oral delivery. Significance and Impact of the Study: Oral vaccination by the B. subtilis vehicle containing VP28 opens a new way for designing practical vaccines to control WSSV.     

Antigenic and immunogenic properties of truncated VP28 protein of white spot syndrome virus in Procambarus clarkii

Previous studies identify VP28 envelope protein of white spot syndrome virus (WSSV) as its main antigenic protein. Although implicated in viral infectivity, its functional role remains unclear. In the current study, we described the production of polyclonal antibodies to recombinant truncated VP28 proteins including deleted N-terminal (rVP28ΔN), C-terminal (rVP28ΔC) and middle (rVP28ΔM). In antigenicity assays, antibodies developed from VP28 truncations lacking the N-terminal or middle regions showed significantly lowered neutralization of WSSV in crayfish, Procambarus clarkii. Further immunogenicity analysis showed reduced relative percent survival (RPS) in crayfish vaccinating with these truncations before challenge with WSSV. These results indicated that N-terminal (residues 1–27) and middle region (residues 35–95) were essential to maintain the neutralizing linear epitopes of VP28 and responsible in eliciting immune response. Thus, it is most likely that these regions are exposed on VP28, and will be useful for rational design of effective vaccines targeting VP28 of WSSV.     

Effect of hyperthermia on the replication of white spot syndrome virus (WSSV) in Procambarus clarkii

The effect of hyperthermia on the development of white spot syndrome virus (WSSV) in the crayfish Procambarus clarkii was studied by competitive PCR. Crayfish were exposed to different temperatures (24 +/- 1 and 32 +/- 1 degrees C) after WSSV injection. No mortality was observed when crayfish were held at 32 +/- 1 degrees C, but mortality reached 100% when crayfish were transferred to 24 +/- 1 degrees C. Competitive PCR showed that viral levels at 32 +/- 1 degrees C remained at 10(5) copies mg(-1) tissue, while at 24 +/- 1 degrees C levels were significantly higher, rising from 10(4) to 10(10) copies mg(-1) tissue. These results suggest that hyperthermia reduces viral replication, but does not eliminate viral particles from WSSV-infected crayfish.     

Protection of Procambarus clarkii against white spot syndrome virus using recombinant oral vaccine expressed in Pichia pastoris

The potential for oral vaccination of crayfish against white spot syndrome virus was investigated. The envelope proteins VP19 and VP28 were expressed in yeast (Pichia pastoris). The expressed proteins were used as oral vaccines in different forms viz., in whole culture form, whole culture sonicated form, whole culture centrifuged supernatant form, and cell residue form. The recombinant proteins were mixed with food pellets and fed to crayfish for 25 days. The vaccinated groups were divided into two even groups and challenged on the 3rd and 21st day of post vaccination. Among different vaccine groups the relative percent survival (RPS) values of sonicated form and supernatant form vaccines were found the best and met the criterion (>RPS 60%) of effective vaccine even after 21st day of post vaccination. Development of vaccine by using recombinant proteins VP19 and VP28 in yeast as expression vector was feasible with significant effects.     

Protection of Procambarus clarkii against white spot syndrome virus using inactivated WSSV

White spot syndrome virus (WSSV) is a highly pathogenic and prevalent virus infecting shrimp and other crustaceans. The potentiality of binary ethylenimine (BEI)-inactivated WSSV against WSSV in crayfish, Procambarus clarkii, was investigated in this study. Efficacy of BEI-inactivated WSSV was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish injected with BEI-inactivated WSSV showed a better survival (P < 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared to the control. Calculated relative percent survival (RPS) values were 77% and 60% on the 7th and 21st dpv for 2 mM BEI-inactivated WSSV, and 63%, 30% on 7th and 21st dpv for 3 mM BEI-inactivated WSSV. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to 3 mM BEI and heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect P. clarkii from WSSV.     

Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori)

Cellular retinoic acid binding protein (CRABP) is a member of intracellular lipid-binding protein (iLBP), and closely associated with retinoic acid (RA) activity. We have cloned the CRABP gene from silkworm pupae and studied the interaction between Bombyx mori CRABP (BmCRABP) and all-trans retinoic acid (atRA). The MTT assay data indicated that when BmCRABP is overexpressed in Bm5 cells, the cells dramatically resisted to atRA-induced growth inhibition. Conversely, the cells were sensitive to atRA treatment upon knocking down the BmCRABP expression. Subcellular localization revealed that BmCRABP is a cytoplasm protein, even when treated with atRA, the CRABP still remained in the cytoplasm. These data demonstrated that the function of BmCRABP have an effect on the physiological function of atRA.     

Characterization of v-sis protein expressed in silkworm larvae using the Bombyx mori nuclear polyhedrosis virus vector

The v-sis oncogene of simian sarcoma virus encodes a protein which is homologous to the human platelet-derived growth factor B-chain. The v-sis protein undergoes a series of processing steps including dimer formation and proteolytic digestion to generate several molecular sizes of the protein. Two of these v-sis proteins were expressed alone or as polyhedrin-sis fusion proteins using the Bombyx mori nuclear polyhedrosis virus vector. The polyhedrin-sis fusion proteins contained a collagenase-sensitive site at the junction. The expression levels of the fusion proteins whose polyhedrin portions consisted of only 8 amino-terminal amino acids were 3-4 times higher than those of non-fusion proteins. One of these fusion proteins was expressed in silkworm larvae and the v-sis protein was isolated from the fusion protein by collagenolysis followed by chromatography. Because the purified v-sis protein exhibited the same molecular size on SDS-polyacrylamide gels under reducing and non-reducing conditions, it was concluded to be monomeric in structure. It possessed chemotactic activity but lacked mitogenic activity. In addition, a small amount (approximately 1%) of monomeric v-sis protein was converted in vitro to the mitogenically active v-sis protein, which could be a homo-dimer.     

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.     

Molecular docking analyses of Avicennia marinaderived phytochemicals against white spot syndrome virus (WSSV) envelope protein-VP28

White spot syndrome (WSS) is one of the most common and most disastrous diseases of shrimp worldwide. It causes up to 100% mortality within 3 to 4 days in commercial shrimp farms, resulting in large economic losses to the shrimp farming industry. VP28 envelope protein of WSSV is reported to play a key role in the systemic infection in shrimps. Considering the most sombre issue of viral disease in cultivated shrimp, the present study was undertaken to substantiate the inhibition potential of Avicennia marinaderived phytochemicals against the WSSV envelope protein VP28. Seven A. marina-derived phytochemicals namely stigmasterol, triterpenoid, betulin, lupeol, avicenol-A, betulinic acid and quercetin were docked against the WSSV protein VP28 by using Argus lab molecular docking software. The chemical structures of the phytochemicals were retrieved from Pubchem database and generated from SMILES notation. Similarly the protein structure of the envelope protein was obtained from protein data bank (PDB-ID: 2ED6). Binding sites were predicted by using ligand explorer software. Among the phytochemicals screened, stigmasterol, lupeol and betulin showed the best binding exhibiting the potential to block VP28 envelope protein of WSSV, which could possibly inhibit the attachment of WSSV to the host species. Further experimental studies will provide a clear understanding on the mode of action of these phytochemicals individually or synergistically against WSSV envelope protein and can be used as an inhibitory drug to reduce white spot related severe complications in crustaceans.