大菱鲆红体病虹彩病毒的流行情况调查及其主要衣壳蛋白在毕赤酵母中的重组表达
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摘要
自2001年以来由大菱鲆红体病虹彩病毒(turbot reddish body iridovirus, TRBIV)引发的大菱鲆病毒性红体病(viral reddish body syndrome of turbot, VRBS)在我国养殖大菱鲆中的流行日益严重,已成为制约着该养殖产业健康可持续发展的最严重的瓶颈因素之一。
本研究利用已建立的PCR检测技术对TRBIV的流行情况进行了调查,调查结果显示:(1) TRBIV的流行已遍布山东半岛沿海地区,苗期、养成期和亲鱼期的大菱鲆均可感染TRBIV,VRBS已成为最严重的大菱鲆疾病之一。(2)养殖水温是影响该病发生的重要因素之一:在养殖水温高于18°C时,患病鱼的死亡率较高、临床症状明显,3个月内的累积死亡率高达90%以上;养殖水温低于18°C时,病毒主要以潜伏状态出现于症状不明显或无明显临床症状的大菱鲆体内,且感染鱼的死亡率较低。(3)调查发现养殖密度也是加重该病发生的因素之一,养殖密度偏高时感染鱼的死亡率明显增加。(4)利用TRBIV的PCR检测技术,发现除肌肉外大菱鲆的脑、鳃、心脏、肝脏、消化道、脾脏、肾脏、血液、性腺等均为TRBIV的敏感组织;(5)在大菱鲆亲鱼的性腺中检测到TRBIV的存在,因此,TRBIV的垂直传播途径需要深入调查和研究。
本研究还根据TRBIV主要衣壳蛋白(Major Capsid Protein, MCP)基因的全序列,利用Omiga2.0及Primer Premier5.0软件对其进行分析并设计其特异性引物。以本实验室构建的克隆质粒pUCm-T/TRBIV MCP为模板,扩增部分为TRBIV MCP全序列中的3 bp到1359 bp之间的区段(即去除了起始密码子ATG,终止密码子TAA后的区段),经PCR扩增得到一条约为1.4 Kb的特异性条带,将经过酶切后的PCR产物、表达载体pGAPZaA定向连接后电转导于E.coli DH5a(recA1 endA1),经过ZeocinTM初筛,PCR和双酶切检测,核酸序列测定后确定成功构建了重组质粒pGAPZaA/TRBIV-MCP。重组质粒pGAPZaA/TRBIV MCP经AvrⅡ酶切线性化后电转导于毕赤酵母X-33中,重组酵母经高浓度的ZeocinTM初筛,提取阳性酵母菌的基因组DNA,经PCR检测和鉴定后确定成功构建了阳性重组毕赤酵母。对阳性酵母小量发酵后,取其上清进行SDS-PAGE电泳检测及用anti-myc-HRP抗体进行Elisa-blot和Westen Blot检测,均检测到目的蛋白。另外又对其表达因素进行了初步探索和优化,为下一步阳性酵母菌的大量发酵,目的蛋白的大量制备,以便为研究TRBIV的宿主相互作用蛋白及TRBIV的免疫检测试剂打下了基础。
Turbot, Scophthalmus maximus, as a traditionally farmed food fish species in the world. Since it was introduced to China by Yellow Sea Fisheries Research Institute in 1992, and cultured vastly in the Jiaodong Peninsula, Shandong Province, from 1998 to now , turbot has become the most important aquaculture species in coastal areas of northern China. However, a novel virus, turbot reddish body iridovirus ( TRBIV ) caused a new kind of epizootic disease“Viral reddish body syndrome”(VRBS) in farmed turbot in 2001, which is belonging to the genus meglocytivirus of the family Iridoviridae, and it has caused severe mortalities of farmed turbot and led to serious economic losses in China since then. Consequently, VRBS was one of the most difficult facters for the sustaining development of turbot culture because there was no any method to control the Propagation of TRBIV in vivo up to now.
The auther investigated the epidemics of TRBIV from 2005 to 2007, basing on the conclusions drew by Prof. Shi Cheng-yin at 2004. Epidemic investigation of TRBIV showed that thousands of infected fish generally lacked external lesions. The only consistent abnormalities were pale gills with local haemorrhages , petechial haemorrhages in fins and fin bases , especially haemorrhages in muscleand skin. Anaemia , pallor and enlarged kidneys were usually observed in severe affected fish. However, there were farmed turbot dead with some new sympotoms, such as: pale body color, an enlarged abdomen, protruding eyes and so on. Investigation also showed that farmed turbot had lower rate of mortality when they lived under water tempreture 18°C. In the epidemic investigation, we also find that higher desity about farmed tubort will aggravate the disease in the farmed turbot.
Using a pair of primers that designed from the highly conserved region of the major capsid protein gene of TRBIV, a PCR product of 781bp was attained with the DNA template of the farmed turbots that had obvious clinical signs of VRBS caused by TRBIV. TRBIV was detected in these tissuses, such as: brain, gill , heart, liver, digestive system , spleen , kidney , blood and the gonad of the parent turbot. However, no virus was detected in the muscle interesting. These results implied that the virus lied vertical transmission through the epidemic investigation. So it is necessary to study vertical transmission of TRBIV by other methods or technologies in the future. But TRBIV was also detected in the turbots, which had pale body color, an enlarged abdomen and protruding eyes, or no any clinical signs about VRBS, and these living under water tempreture 18°C. These results implies that TRBIV can hide in the body of tubort and and destroy the immune system of turbot, lastly result in the death of infected turbot with the accumulation of virus particles.
In this study, we designed PCR primers which eliminate the signal pepetide in order to be expressed properly. With the vector pUCm-T/TRBIV MCP as template, we can get the PCR products TRBIV MCP 1373 bp. After digestion with endonuclease, the PCR products were ligated into eukaryotic expression vector pGAPZαA, and then transformed into E.coli Top10F’by electroporation, and the recombinant vectors pGAPZαA/TRBIV MCP were achieved.
After the recombinant vectors were corrected by PCR, digestion with endonuclease, and sequence, the recombinant vector pGAPZαA/TRBIV MCP was digested by AvrⅡand transformed into Pichia pastoris X-33 by electroporation. At first, ZeocinTM was used to screen the recombinant yeast clones. Secondly the recombinant yeasts were identified by PCR with the recombinant yeast genosome as template. Then the higher expressional recombinant yeasts were detected by Elisa-blot. We picked out seven of the identified yeasts to culture at 30°C, and ferment at 28°C, and collect the supernatant by centrifuge lastly, Then the total proteins were obtained. The above proteins were detected by SDS-PAGE and Western-blot, and a protein at the size of 54KD was signal showed. That result implied that the recombinant TRBIV MCP was expressedsuccessfully.
本研究利用已建立的PCR检测技术对TRBIV的流行情况进行了调查,调查结果显示:(1) TRBIV的流行已遍布山东半岛沿海地区,苗期、养成期和亲鱼期的大菱鲆均可感染TRBIV,VRBS已成为最严重的大菱鲆疾病之一。(2)养殖水温是影响该病发生的重要因素之一:在养殖水温高于18°C时,患病鱼的死亡率较高、临床症状明显,3个月内的累积死亡率高达90%以上;养殖水温低于18°C时,病毒主要以潜伏状态出现于症状不明显或无明显临床症状的大菱鲆体内,且感染鱼的死亡率较低。(3)调查发现养殖密度也是加重该病发生的因素之一,养殖密度偏高时感染鱼的死亡率明显增加。(4)利用TRBIV的PCR检测技术,发现除肌肉外大菱鲆的脑、鳃、心脏、肝脏、消化道、脾脏、肾脏、血液、性腺等均为TRBIV的敏感组织;(5)在大菱鲆亲鱼的性腺中检测到TRBIV的存在,因此,TRBIV的垂直传播途径需要深入调查和研究。
本研究还根据TRBIV主要衣壳蛋白(Major Capsid Protein, MCP)基因的全序列,利用Omiga2.0及Primer Premier5.0软件对其进行分析并设计其特异性引物。以本实验室构建的克隆质粒pUCm-T/TRBIV MCP为模板,扩增部分为TRBIV MCP全序列中的3 bp到1359 bp之间的区段(即去除了起始密码子ATG,终止密码子TAA后的区段),经PCR扩增得到一条约为1.4 Kb的特异性条带,将经过酶切后的PCR产物、表达载体pGAPZaA定向连接后电转导于E.coli DH5a(recA1 endA1),经过ZeocinTM初筛,PCR和双酶切检测,核酸序列测定后确定成功构建了重组质粒pGAPZaA/TRBIV-MCP。重组质粒pGAPZaA/TRBIV MCP经AvrⅡ酶切线性化后电转导于毕赤酵母X-33中,重组酵母经高浓度的ZeocinTM初筛,提取阳性酵母菌的基因组DNA,经PCR检测和鉴定后确定成功构建了阳性重组毕赤酵母。对阳性酵母小量发酵后,取其上清进行SDS-PAGE电泳检测及用anti-myc-HRP抗体进行Elisa-blot和Westen Blot检测,均检测到目的蛋白。另外又对其表达因素进行了初步探索和优化,为下一步阳性酵母菌的大量发酵,目的蛋白的大量制备,以便为研究TRBIV的宿主相互作用蛋白及TRBIV的免疫检测试剂打下了基础。
Turbot, Scophthalmus maximus, as a traditionally farmed food fish species in the world. Since it was introduced to China by Yellow Sea Fisheries Research Institute in 1992, and cultured vastly in the Jiaodong Peninsula, Shandong Province, from 1998 to now , turbot has become the most important aquaculture species in coastal areas of northern China. However, a novel virus, turbot reddish body iridovirus ( TRBIV ) caused a new kind of epizootic disease“Viral reddish body syndrome”(VRBS) in farmed turbot in 2001, which is belonging to the genus meglocytivirus of the family Iridoviridae, and it has caused severe mortalities of farmed turbot and led to serious economic losses in China since then. Consequently, VRBS was one of the most difficult facters for the sustaining development of turbot culture because there was no any method to control the Propagation of TRBIV in vivo up to now.
The auther investigated the epidemics of TRBIV from 2005 to 2007, basing on the conclusions drew by Prof. Shi Cheng-yin at 2004. Epidemic investigation of TRBIV showed that thousands of infected fish generally lacked external lesions. The only consistent abnormalities were pale gills with local haemorrhages , petechial haemorrhages in fins and fin bases , especially haemorrhages in muscleand skin. Anaemia , pallor and enlarged kidneys were usually observed in severe affected fish. However, there were farmed turbot dead with some new sympotoms, such as: pale body color, an enlarged abdomen, protruding eyes and so on. Investigation also showed that farmed turbot had lower rate of mortality when they lived under water tempreture 18°C. In the epidemic investigation, we also find that higher desity about farmed tubort will aggravate the disease in the farmed turbot.
Using a pair of primers that designed from the highly conserved region of the major capsid protein gene of TRBIV, a PCR product of 781bp was attained with the DNA template of the farmed turbots that had obvious clinical signs of VRBS caused by TRBIV. TRBIV was detected in these tissuses, such as: brain, gill , heart, liver, digestive system , spleen , kidney , blood and the gonad of the parent turbot. However, no virus was detected in the muscle interesting. These results implied that the virus lied vertical transmission through the epidemic investigation. So it is necessary to study vertical transmission of TRBIV by other methods or technologies in the future. But TRBIV was also detected in the turbots, which had pale body color, an enlarged abdomen and protruding eyes, or no any clinical signs about VRBS, and these living under water tempreture 18°C. These results implies that TRBIV can hide in the body of tubort and and destroy the immune system of turbot, lastly result in the death of infected turbot with the accumulation of virus particles.
In this study, we designed PCR primers which eliminate the signal pepetide in order to be expressed properly. With the vector pUCm-T/TRBIV MCP as template, we can get the PCR products TRBIV MCP 1373 bp. After digestion with endonuclease, the PCR products were ligated into eukaryotic expression vector pGAPZαA, and then transformed into E.coli Top10F’by electroporation, and the recombinant vectors pGAPZαA/TRBIV MCP were achieved.
After the recombinant vectors were corrected by PCR, digestion with endonuclease, and sequence, the recombinant vector pGAPZαA/TRBIV MCP was digested by AvrⅡand transformed into Pichia pastoris X-33 by electroporation. At first, ZeocinTM was used to screen the recombinant yeast clones. Secondly the recombinant yeasts were identified by PCR with the recombinant yeast genosome as template. Then the higher expressional recombinant yeasts were detected by Elisa-blot. We picked out seven of the identified yeasts to culture at 30°C, and ferment at 28°C, and collect the supernatant by centrifuge lastly, Then the total proteins were obtained. The above proteins were detected by SDS-PAGE and Western-blot, and a protein at the size of 54KD was signal showed. That result implied that the recombinant TRBIV MCP was expressedsuccessfully.
引文
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