对虾白斑病毒（WSSV）囊膜蛋白蛋黄抗体及凋亡抑制基因ORF390R的研究
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摘要
对虾白斑综合症病毒(White Spot Syndrome Virus,WSSV)是一种具有囊膜的双链DNA病毒,是造成自1993年以来中国、东南亚及美洲等世界范围内大面积暴发对虾病毒性疾病的主要病原。该病传播速度快,感染虾群在3～7d内死亡率高达90～100%,对我国及世界的对虾养殖造成了严重的危害,而目前尚未有有效的防治方法。
本研究对水体温度在WSSV感染螯虾中起何种影响做了初步的研究。将螯虾培养在不同温度的水体中,通过注射来感染WSSV来观察死亡率的变化。经过RT-PCR以及组织切片等实验,说明低温可以影响WSSV在螯虾体内的繁殖,病毒在低温下可以在宿主体内长期存活。
用PCR的方法将四种囊膜蛋白基因扩增出来并克隆到pMAL-C2原核表达载体中。将表达的麦芽糖融合蛋白通过亲和层析的方法纯化出来,注射母鸡来产生对应的抗体。克氏螯虾的抗体中合实验结果显示,囊膜蛋白VP28与VP19的可以中合WSSV对螯虾的感染,而且这种综合是通过体内注射是浓度依赖性的。相关囊膜蛋白鸡抗的口服也可以作为一种潜在的免疫治疗方法来阻止病毒在宿主的感染。囊膜蛋白的口服注射免疫实验证明,在螯虾体内存在着类似于哺乳动物的免疫系统,外源刺激物可以引起相应的免疫反应,以增强对病原体的抵御。
由于到目前为止,还没有稳定的传代细胞能有效的接种和繁殖细胞,使得对虾白斑病毒基因组得研究进展缓慢,使得对病毒感染得分子机制不太明了,对病毒感染对虾也没有有效得防治方法。本文借鉴杆状病毒得研究方法,在昆虫细胞内对WSSV的凋亡抑制基因进行了鉴定,并对其进行了原核与真核的表达。利用
White spot disease (WSD) is at present the most serious viral disease for cultivated shrimp, which caused very serious and widespread losses in the shrimp industry around the world. The causative agent is commonly known as white spot syndrome virus (WSSV) owing to the main clinic sign 'white spots' in the exo-mesoderm under the carapace.The effect of water temperature on the development of white spot disease was studied. Rayfish were exposed to different temperatures (4℃, 16℃ and 25℃) after WSSV injection and the mortalities were recorded over a period of 30 days. The results clearly show that low temperature affects the WSSV pathogenicity in cryfish. Moreover, total haemocyte and granular cell counts of cryfish held at different temperatures were not significant different. The phenoloxidase activities in haemocyte lysate supernant (HLS) of crayfish at all temperature group remain similar. It is concluded that low temperature could prevent WSSV proliferation and crayfish might act as a carrier of WSSV at low temperature.The four envelope protein genes were constructed into pMAL-c2 prokaryotic expression vector and the maltose-fusion expression proteins were purified. The fusion proteins were used as immugens with chickens to generate antibodies. Results of freshwater crayfish neutralization experiment suggested that VP28 and VP19 chicken antibodies were able to neutralize WSSV infection of freshwater crayfish in a
concentration dependent manner upon intramuscular infection. The freshwater crayfishes were firstly fed WSSV infected meal and then fed with basal diet or diet supplement with VP28 and VP19 chicken antibodies. All the freshwater crayfish died fed with basal diet at 30 days. Conversely, the survival rates of freshwater crayfish fed with the diet containing VP28 and VP19 chicken antibodies can be reduced to 60% and 50%, respectively. Oral passive immunization experiment showed that fed crayfish with diet supplement with chicken antibodies could prevent the WSSV infection in a certain extent. All results showed that VP28 and VP19 are likely to play key roles in the systemic WSSV infection in freshwater crayfish and the oral passive immunization of chicken antibodies has potential for immunotherapeutic application to prevent WSSV infection.In this study, we confirmed that the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPV. 35k/pol+ lacks a functional p35 gene undergoing apoptosis and its infection could induce Sf9 cells apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPV. p35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPV. p35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anliapoptotic gene. The ORF displays two putative caspase9cleavage sites LLVETDGPS , VKLEHDGSK , a caspase3 cleavage site EEDEVDGVP . The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells didn't show obvious characteristics of apoptosis when infected with AcMNPV. p35k/pol+. And the transient expression of ORF390 allowed AcMNPV. p35k/pol+ replication in Sf9 cells and result in formation of polyhedra successfully. The results indicate that function of ORF 390 in WSSV is a kind of apoplotic suppressor like P35 in AcMNPV.
本研究对水体温度在WSSV感染螯虾中起何种影响做了初步的研究。将螯虾培养在不同温度的水体中,通过注射来感染WSSV来观察死亡率的变化。经过RT-PCR以及组织切片等实验,说明低温可以影响WSSV在螯虾体内的繁殖,病毒在低温下可以在宿主体内长期存活。
用PCR的方法将四种囊膜蛋白基因扩增出来并克隆到pMAL-C2原核表达载体中。将表达的麦芽糖融合蛋白通过亲和层析的方法纯化出来,注射母鸡来产生对应的抗体。克氏螯虾的抗体中合实验结果显示,囊膜蛋白VP28与VP19的可以中合WSSV对螯虾的感染,而且这种综合是通过体内注射是浓度依赖性的。相关囊膜蛋白鸡抗的口服也可以作为一种潜在的免疫治疗方法来阻止病毒在宿主的感染。囊膜蛋白的口服注射免疫实验证明,在螯虾体内存在着类似于哺乳动物的免疫系统,外源刺激物可以引起相应的免疫反应,以增强对病原体的抵御。
由于到目前为止,还没有稳定的传代细胞能有效的接种和繁殖细胞,使得对虾白斑病毒基因组得研究进展缓慢,使得对病毒感染得分子机制不太明了,对病毒感染对虾也没有有效得防治方法。本文借鉴杆状病毒得研究方法,在昆虫细胞内对WSSV的凋亡抑制基因进行了鉴定,并对其进行了原核与真核的表达。利用
White spot disease (WSD) is at present the most serious viral disease for cultivated shrimp, which caused very serious and widespread losses in the shrimp industry around the world. The causative agent is commonly known as white spot syndrome virus (WSSV) owing to the main clinic sign 'white spots' in the exo-mesoderm under the carapace.The effect of water temperature on the development of white spot disease was studied. Rayfish were exposed to different temperatures (4℃, 16℃ and 25℃) after WSSV injection and the mortalities were recorded over a period of 30 days. The results clearly show that low temperature affects the WSSV pathogenicity in cryfish. Moreover, total haemocyte and granular cell counts of cryfish held at different temperatures were not significant different. The phenoloxidase activities in haemocyte lysate supernant (HLS) of crayfish at all temperature group remain similar. It is concluded that low temperature could prevent WSSV proliferation and crayfish might act as a carrier of WSSV at low temperature.The four envelope protein genes were constructed into pMAL-c2 prokaryotic expression vector and the maltose-fusion expression proteins were purified. The fusion proteins were used as immugens with chickens to generate antibodies. Results of freshwater crayfish neutralization experiment suggested that VP28 and VP19 chicken antibodies were able to neutralize WSSV infection of freshwater crayfish in a
concentration dependent manner upon intramuscular infection. The freshwater crayfishes were firstly fed WSSV infected meal and then fed with basal diet or diet supplement with VP28 and VP19 chicken antibodies. All the freshwater crayfish died fed with basal diet at 30 days. Conversely, the survival rates of freshwater crayfish fed with the diet containing VP28 and VP19 chicken antibodies can be reduced to 60% and 50%, respectively. Oral passive immunization experiment showed that fed crayfish with diet supplement with chicken antibodies could prevent the WSSV infection in a certain extent. All results showed that VP28 and VP19 are likely to play key roles in the systemic WSSV infection in freshwater crayfish and the oral passive immunization of chicken antibodies has potential for immunotherapeutic application to prevent WSSV infection.In this study, we confirmed that the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPV. 35k/pol+ lacks a functional p35 gene undergoing apoptosis and its infection could induce Sf9 cells apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPV. p35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPV. p35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anliapoptotic gene. The ORF displays two putative caspase9cleavage sites LLVETDGPS , VKLEHDGSK , a caspase3 cleavage site EEDEVDGVP . The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells didn't show obvious characteristics of apoptosis when infected with AcMNPV. p35k/pol+. And the transient expression of ORF390 allowed AcMNPV. p35k/pol+ replication in Sf9 cells and result in formation of polyhedra successfully. The results indicate that function of ORF 390 in WSSV is a kind of apoplotic suppressor like P35 in AcMNPV.
引文
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