AsiaⅠ型口蹄疫病毒空衣壳抗原的表达与免疫研究
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摘要
FMD是一种严重危害畜牧业生产的烈性传染病。2005年在我国数省区爆发的AsiaⅠ型FMD对国家造成了巨大的经济损失。然而AsiaⅠ型FMDV研究资料相对较少，不利于防控工作的开展。因此本研究选用AsiaⅠ/JQ株为研究材料，对其编码区序列进行了测定，并借助计算机软件进行了生物信息学分析。在此基础上，利用家蚕杆状病毒表达系统表达了空衣壳抗原，并对表达产物进行了免疫效力评估。
(1)FMDV AsiaⅠ/JQ株的编码区序列测定：通过RT-PCR方法获得了AsiaⅠ/JQ株的编码区序列，并应用DNAStar软件进行了生物信息学分析。通过与国内外典型代表毒株进行比较与分析，结果显示该毒株结构蛋白编码区与AisaⅠ型参考毒株同一区域同源性较高，而非结构蛋白编码区L、P2、P3却与O型参考毒株同一区域同源性较高。从利用P1蛋白编码区绘制的系统进化树分析，AsiaⅠ/JQ株与起源于印度的两株AsiaⅠ型病毒遗传距离较近，而利用非编码区绘制的系统进化树却表明与O型的两株病毒遗传距离较近。据此推测该毒株为一株O型与AsiaⅠ型FMDV的重组毒株。
(2)运用Garnier-Robson方法、Chou-Farplus方法和Karplus-Schulz方法预测了AsiaⅠ/JQ株FMDV结构蛋白的二级结构。运用Kyte-Doolittle方法对结构蛋白的亲水性进行分析，用Emini方法预测结构蛋白的表面可及性，以Jameson-Wolf方法预测了结构蛋白的抗原指数。然后综合评价了AsiaⅠ/JQ株结构蛋白的B淋巴细胞表位。结果表明VP1、VP2、VP3三个结构蛋白上存在较多潜在的B细胞表位，VP4上也存在着少量的抗原表位。这一结果提示在设计FMDV基因工程疫苗时应予以综合考虑，不应局限于VP1蛋白。
(3)将AsiaⅠ/JQ株全长6.9Kb的ORF克隆到家蚕杆状病毒转移载体pVL1393中，构建重组家蚕杆状病毒转移质粒pVL-ORF。pVL-ORF与线性化的亲本病毒Bm-BacPAK6 DNA共转染家蚕细胞，经空斑筛选后成功获得了携带有FMDV全长ORF的重组病rBmNPV(ORF)。通过免疫荧光技术证明重组病毒能够正确表达插入的外源基因。将获得的重组病毒rBmNPV(ORF)感染家蚕5龄起幼虫，通过双抗体夹心ELISA法和电镜观察证明目的基因在蚕体中获得较高水平表达并组装成空衣壳。用蚕表达产物作抗原制备疫苗免疫牛，免疫动物均产生了特异性抗体。免疫后21天进行病毒攻击保护试验，获得了2/4的保护率。
(4)采用类似的方法构建了包括AsiaⅠ/JQ株P1-2A和3C基因的重组病毒rBmNPV(P1-2A3C)。通过免疫荧光技术证明重组病毒能够正确表达插入的外源基因。双抗体ELISA法测定目的蛋白在蚕体上获得了极高的表达量。通过电子显微镜技术，在蚕血淋巴中观测到大量的病毒空衣壳。将表达产物制备成疫苗一次免疫牛后，实验动物产生了针对FMDV的特异性抗体。28天后使用10,000BID_(50)同源强毒攻击，获得了4/5的保护率。
(5)参照OIE规定的PD_(50)测定法进行了rBmNPV(P1-2A3C)表达产物的免疫效力实验。结果显示以稀释40倍抗原制备的疫苗其PD_(50)值为3.60，以稀释30倍的抗原制备的疫苗其PD_(50)值为6.34。这一结果为利用家蚕杆状病毒生产的空衣壳疫苗最终走向市场奠定了良好的前期工作基础。
Foot-and-mouth disease (FMD) is one of the most important known pathogens of livestock. In 2005, the disease caused by foot-and-mouth disease (FMDV) serotype Asia I broke out in part of China. But the available research data of type AsiaⅠis relative lack. In this research, we have sequenced the intact open reading frame (ORF) of FMDV AsiaⅠ/JQ strain, and expressed the empty capsids antigen using silkworm- baculovirus expression system.
1. The intact ORF sequence of AsiaⅠ/JQ strain was determined. It is 6987nt long and can be divided into four parts of L, P1, P2 and P3. With the help of the DNAStar ware, these four parts of AsiaⅠ/JQ strain were compared with 7 reference strains. The comparison of the P1 confirmed that AsiaⅠ/JQ has a high identity with type AsiaⅠreference sequences. But the identities of nonstructural proteins coding region were higher with type O strains than type Asia I strains. The phylogentic tree obtained from the nucleotide sequence of P1 gene from AsiaⅠ/JQ strain and 7 reference strains shows that the AsiaⅠ/JQ strain has a close relationship with the type Asia I strains. The phylogentic trees obtained from the nucleotide sequence of nonstructural protein coding regions show that the AsiaⅠ/JQ strain has a close relationship with the type O stains. The results suggest that the gene recombination had occurred between 0 type strain and AsiaⅠtype strain to recombine the AsiaⅠ/JQ strain.
2. The secondary structure of capid protein of AsiaⅠ/JQ strain was predicted by the methods of Garnier-Robson, Chou-Farplus and Karplus-Schulzbased, and hydrophilicity plot, surface probability plot and antigenic index were obtained by the methods of Kyte-doolittle, Emini and Jameson-Wolf respectively. Combining the resultes according to these methods, the B cell epitopes of capsid protein of FMDV were predicted. The results showed that there is much flexible region such as coil region and turn region in capsid protein of FMDV, and more B cell epitopes in VP1, VP2 and VP3 than VP4. This study would be helpful for identification of B cell epitopes for capsid protein using experimental methods and research of reverse vaccine of FMDV.
3. The intact ORF of foot-and-mouth disease virus (FMDV) AsiaⅠ/JQ strain was inserted into the transfer vector pVL1393 to generate plasmid pVL-ORF. The recombinant silkworm baculovirus rBmNPV(ORF) containing the intact ORF of FMDV was obtained after co-transfection and screening. Indirect immunofluore- scence test was used to verify that rBamNPV(ORF) could validly express the target cassette in Bm-N cell. The early 5th instar larvae of silkworm were infected with the recombinant virus. The polyprotein of FMDV expressed in silkworm was confirmed by sandwich-ELISA and empty capsid like particles could be observed in haemolymph. Expression products from silkworm were used as antigen to immunize the cattle. The specific antibody was induced in all vaccinated animals. Challenged with 10,000 BID_(50) of virulent homologous virus, two of the four were completely protected, and clinical symptoms were alleviated and delayed in the others.
4. The recombinant silkworm baculovirus rBamNPV(P1-2A3C) which contained the intact P1-2A and 3C protease coding regions of AsiaⅠ/JQ stain was obtained. Indirect immunofluorescence test was used to verify that rBmNPV(Pl-2A3C)could validly express the target cassette. The sandwich-ELISA confirmed that the quantity of FMDV antigen in haemolymph was about 300 microgramme or more. Expression products from silkworm were used as antigen to immunize the cattle. The specific antibody was induced in all vaccinated animals. Challenged with virulent homologous virus, four of the five were completely protected, and clinical symptoms were alleviated and delayed in the other one.
5. We followed the bovine potency test protocol described by the OIE to test this empty capsids vaccine potency. The PD50 (50 % protective dose) potency were detected on cattle with 10,000 BID_(50) of virulent homologous virus. The results showed the vaccine potency of the batch immunized with the expressed antigens diluted 40 folds could get 3.60 PD_(50) per dose and the vaccine potency of the batch with the expressed antigens diluted 30 folds could get 6.34 PD_(50) per dose. These experiments lead to a conclusion that it is feasible to make use of silkworm- baculovirus expression system for FMD vaccine production.
(1)FMDV AsiaⅠ/JQ株的编码区序列测定：通过RT-PCR方法获得了AsiaⅠ/JQ株的编码区序列，并应用DNAStar软件进行了生物信息学分析。通过与国内外典型代表毒株进行比较与分析，结果显示该毒株结构蛋白编码区与AisaⅠ型参考毒株同一区域同源性较高，而非结构蛋白编码区L、P2、P3却与O型参考毒株同一区域同源性较高。从利用P1蛋白编码区绘制的系统进化树分析，AsiaⅠ/JQ株与起源于印度的两株AsiaⅠ型病毒遗传距离较近，而利用非编码区绘制的系统进化树却表明与O型的两株病毒遗传距离较近。据此推测该毒株为一株O型与AsiaⅠ型FMDV的重组毒株。
(2)运用Garnier-Robson方法、Chou-Farplus方法和Karplus-Schulz方法预测了AsiaⅠ/JQ株FMDV结构蛋白的二级结构。运用Kyte-Doolittle方法对结构蛋白的亲水性进行分析，用Emini方法预测结构蛋白的表面可及性，以Jameson-Wolf方法预测了结构蛋白的抗原指数。然后综合评价了AsiaⅠ/JQ株结构蛋白的B淋巴细胞表位。结果表明VP1、VP2、VP3三个结构蛋白上存在较多潜在的B细胞表位，VP4上也存在着少量的抗原表位。这一结果提示在设计FMDV基因工程疫苗时应予以综合考虑，不应局限于VP1蛋白。
(3)将AsiaⅠ/JQ株全长6.9Kb的ORF克隆到家蚕杆状病毒转移载体pVL1393中，构建重组家蚕杆状病毒转移质粒pVL-ORF。pVL-ORF与线性化的亲本病毒Bm-BacPAK6 DNA共转染家蚕细胞，经空斑筛选后成功获得了携带有FMDV全长ORF的重组病rBmNPV(ORF)。通过免疫荧光技术证明重组病毒能够正确表达插入的外源基因。将获得的重组病毒rBmNPV(ORF)感染家蚕5龄起幼虫，通过双抗体夹心ELISA法和电镜观察证明目的基因在蚕体中获得较高水平表达并组装成空衣壳。用蚕表达产物作抗原制备疫苗免疫牛，免疫动物均产生了特异性抗体。免疫后21天进行病毒攻击保护试验，获得了2/4的保护率。
(4)采用类似的方法构建了包括AsiaⅠ/JQ株P1-2A和3C基因的重组病毒rBmNPV(P1-2A3C)。通过免疫荧光技术证明重组病毒能够正确表达插入的外源基因。双抗体ELISA法测定目的蛋白在蚕体上获得了极高的表达量。通过电子显微镜技术，在蚕血淋巴中观测到大量的病毒空衣壳。将表达产物制备成疫苗一次免疫牛后，实验动物产生了针对FMDV的特异性抗体。28天后使用10,000BID_(50)同源强毒攻击，获得了4/5的保护率。
(5)参照OIE规定的PD_(50)测定法进行了rBmNPV(P1-2A3C)表达产物的免疫效力实验。结果显示以稀释40倍抗原制备的疫苗其PD_(50)值为3.60，以稀释30倍的抗原制备的疫苗其PD_(50)值为6.34。这一结果为利用家蚕杆状病毒生产的空衣壳疫苗最终走向市场奠定了良好的前期工作基础。
Foot-and-mouth disease (FMD) is one of the most important known pathogens of livestock. In 2005, the disease caused by foot-and-mouth disease (FMDV) serotype Asia I broke out in part of China. But the available research data of type AsiaⅠis relative lack. In this research, we have sequenced the intact open reading frame (ORF) of FMDV AsiaⅠ/JQ strain, and expressed the empty capsids antigen using silkworm- baculovirus expression system.
1. The intact ORF sequence of AsiaⅠ/JQ strain was determined. It is 6987nt long and can be divided into four parts of L, P1, P2 and P3. With the help of the DNAStar ware, these four parts of AsiaⅠ/JQ strain were compared with 7 reference strains. The comparison of the P1 confirmed that AsiaⅠ/JQ has a high identity with type AsiaⅠreference sequences. But the identities of nonstructural proteins coding region were higher with type O strains than type Asia I strains. The phylogentic tree obtained from the nucleotide sequence of P1 gene from AsiaⅠ/JQ strain and 7 reference strains shows that the AsiaⅠ/JQ strain has a close relationship with the type Asia I strains. The phylogentic trees obtained from the nucleotide sequence of nonstructural protein coding regions show that the AsiaⅠ/JQ strain has a close relationship with the type O stains. The results suggest that the gene recombination had occurred between 0 type strain and AsiaⅠtype strain to recombine the AsiaⅠ/JQ strain.
2. The secondary structure of capid protein of AsiaⅠ/JQ strain was predicted by the methods of Garnier-Robson, Chou-Farplus and Karplus-Schulzbased, and hydrophilicity plot, surface probability plot and antigenic index were obtained by the methods of Kyte-doolittle, Emini and Jameson-Wolf respectively. Combining the resultes according to these methods, the B cell epitopes of capsid protein of FMDV were predicted. The results showed that there is much flexible region such as coil region and turn region in capsid protein of FMDV, and more B cell epitopes in VP1, VP2 and VP3 than VP4. This study would be helpful for identification of B cell epitopes for capsid protein using experimental methods and research of reverse vaccine of FMDV.
3. The intact ORF of foot-and-mouth disease virus (FMDV) AsiaⅠ/JQ strain was inserted into the transfer vector pVL1393 to generate plasmid pVL-ORF. The recombinant silkworm baculovirus rBmNPV(ORF) containing the intact ORF of FMDV was obtained after co-transfection and screening. Indirect immunofluore- scence test was used to verify that rBamNPV(ORF) could validly express the target cassette in Bm-N cell. The early 5th instar larvae of silkworm were infected with the recombinant virus. The polyprotein of FMDV expressed in silkworm was confirmed by sandwich-ELISA and empty capsid like particles could be observed in haemolymph. Expression products from silkworm were used as antigen to immunize the cattle. The specific antibody was induced in all vaccinated animals. Challenged with 10,000 BID_(50) of virulent homologous virus, two of the four were completely protected, and clinical symptoms were alleviated and delayed in the others.
4. The recombinant silkworm baculovirus rBamNPV(P1-2A3C) which contained the intact P1-2A and 3C protease coding regions of AsiaⅠ/JQ stain was obtained. Indirect immunofluorescence test was used to verify that rBmNPV(Pl-2A3C)could validly express the target cassette. The sandwich-ELISA confirmed that the quantity of FMDV antigen in haemolymph was about 300 microgramme or more. Expression products from silkworm were used as antigen to immunize the cattle. The specific antibody was induced in all vaccinated animals. Challenged with virulent homologous virus, four of the five were completely protected, and clinical symptoms were alleviated and delayed in the other one.
5. We followed the bovine potency test protocol described by the OIE to test this empty capsids vaccine potency. The PD50 (50 % protective dose) potency were detected on cattle with 10,000 BID_(50) of virulent homologous virus. The results showed the vaccine potency of the batch immunized with the expressed antigens diluted 40 folds could get 3.60 PD_(50) per dose and the vaccine potency of the batch with the expressed antigens diluted 30 folds could get 6.34 PD_(50) per dose. These experiments lead to a conclusion that it is feasible to make use of silkworm- baculovirus expression system for FMD vaccine production.
引文
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