表达ZJ-1株新城疫病毒F基因重组鸡痘病毒的筛选及其免疫效力试验
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摘要
新城疫(ND)是由新城疫病毒(NDV)引起的禽类的病毒性传染病之一，广泛流行于世界上许多国家和地区，给养禽业造成了巨大的经济损失。NDV属副粘病毒属，其病毒囊膜由两种蛋白组成，其中之一为融合蛋白(Fusion，F)，它参与病毒的融合与穿透，在疾病的免疫保护方面发挥着重要作用。我国目前仍以疫苗接种为主来控制该病，常用的油乳剂灭活苗和弱毒活苗对该病的控制起到重要作用，但其存在生产成本高，易散毒，使用后干扰ND疫情监测等缺点，因此，有必要研制新一代安全高效的基因工程疫苗。本实验利用本室分离的鹅源ZJ-1株NDV构建了表达F基因的4株重组鸡痘病毒(rFPV_(282E4)-SF_A、rFPV_(LP)-SF_A、rFPV_(282E4)-SF_B和rFPV_(LP)-SF_B)，并对其进行遗传稳定性分析和免疫效力试验。
1 表达ZJ-1株NDV F基因重组鸡痘病毒的筛选及其遗传稳定性测定
利用FuGEN~(TM)6 Transfection Reagent进行转染，分别将含有NDV F基因的转移载体P11SF和PN11SF与282E4株和大空斑鸡痘病毒共转染鸡胚成纤维细胞(CEF)，通过蓝斑筛选，得到纯化的重组病毒rFPV_(282E4)-SF_A、rFPV_(LP)-SF_A、rFPV_(282E4)-SF_B和rFPV_(LP)-SF_B。PCR试验表明重组病毒基因组中含有F基因；间接免疫荧光试验(IFA)表明重组病毒中的F基因能够得到表达。
重组病毒经CEF连续传20代，覆盖含x-gal的琼脂，其在CEF上形成的空斑均为蓝斑。间接免疫荧光试验表明，重组病毒传20代后F基因的表达未受影响。取0、10和20代重组病毒DNA为模板进行PCR，均可扩增出约1.7 Kb的F基因片段。取rFPV_(282E4)-SF_B和rFPV_(LP)-SF_B的0、10和20代PCR产物进行测序后与转移载体中的F基因相比，核苷酸和氨基酸无任何变化，显示了该重组病毒具有良好的遗传稳定性。
2 重组病毒的动物试验
2．1 重组病毒对SPF鸡的免疫效力
5日龄SPF鸡随机分为6组，进行皮下接种W_1FPV_(LP)、rFPV_(282E4)-SF_A、
扬州大学硕士学位论文
rFPvL产SF^、亦PvZ:2以一SF。和:FPvLP一SFB lo4PFUz只，油苗为o.Zml/只，免疫后3
周龄用1护ELDS。F48E8株NDv进行滴鼻攻击。通过EusA抗体水平和保护效率
来评价重组病毒的免疫效力，结果除wtFPvLP组全部死亡外，其余组均100%保护。
2.2重组病毒对商品鸡的免疫效力
将26日龄商品鸡随机分为7组，以105PFu/只的量分别将wtFPvLP、
rFpVZ:2E4一sF^、rFpVLP一sFA、rFpVZsZE4一sF。和亦PVL产sF。进行皮下接种，油苗为
o.Zmlz只，另外设空白对照组。免疫后21天以lo5ELnso F4sEs株Nnv滴鼻攻毒，
观察14天，其保护效率rFPVZsZE4一SF^、rFPVLP一SF^、rFPVZsZE4一SFB和:FPvLP一SFs
分别为64%、60%、52%、88%;油苗和WtFPVLP分别为1 00%、0。
3应用间接ELIsA检测鸡痘病毒抗体方法的初步建立
应用CEF扩增的鸡痘病毒(FPV)提纯后作为抗原建立了具有较高特异性和
灵敏性的间接ELIsA方法来检测FPV抗体。通过方阵滴定法来确定抗原最佳包被
浓度为2.7u留孔，待检血清最佳稀释倍数为1:100，其阳性临界值为OD妻0.098。
将400份FPV或者重组鸡痘病毒免疫过的实验鸡血清用本方法进行检测，其阳性
检出率为81.25%(325/4 00)。此外，将该方法与琼脂扩散试验进行比较检测血清
样品，结果显示本方法的灵敏度比琼脂扩散试验灵敏400一800倍，而且还有特异性
强，操作简便、快速等优点。
综上所述，本试验成功筛选了4株重组鸡痘病毒，并且具有良好的遗传稳定
性，对SPF鸡能提供100%的保护。该疫苗的研制成功将会加速新型基因工程疫苗
替代传统灭活苗的步伐，并将为ND的防制带来新的技术手段。
Newcastle disease (ND) that caused by Newcastle disease virus (NDV) is one of the most important avian diseases. It is widespread in many country and areas throughout the world, and can cause severe economic losses in the poultry industry. NDV is a paramyxovirus possessing two major glycoprotein components of the virion envelope, one of which is the Fusion (F) protein. It is involved in membrane fusion and virus penetration, and is known to play an important role in immunity to the disease. In order to develop new genetic engineering vaccines which are safe and effective, we constructed four recombinant fowlpox virus (rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4 -SFB, rFPVLp-SFB) expressing F gene of ZJ-1 strain of NDV and evaluated its genetic stability and protective efficacy in this study.
1 Selection of recombinant fowlpox viruses (rFPVs) expressing F gene of NDV and their genetic stability
The transfer vectors P11SF and PN11SF were transfected by Fugend? Transfection Reagent mediated transfection on CEF infected with parent 282E4 and large plaque strain FPV. The four rFPVs ( rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVLp-SFB) were selected and purified by blue plaques expressing 3 -galactosidase. The insertion and expression of F genes in rFPVs were confirmed by PCR and IFA respectively.
Four rFPVs were serially passaged to the 20th on CEF monolayers, the expression of F genes were indentified by IFA. The F genes of passages 0, 10, and 20 were amplified by PCR and sequenced. There were no changes on the nucleotide and amino acid. The results showed that the four rFPVs were genetically stable for at least 20
passages.
2 Vaccination trials with rFPVs in chickens
2.1 Protective efficacy of the rFPVs in SPF chickens
Six groups of 5-day-old SPF chickens were vaccinated with Wt-FPVLp, rFPV282E4 -SFA, rFPVtp-SFA, rFPV282E4-SFB, rFPVtp-SFe and inactivated vaccine in oil emulsion by SC route in the nape of neck at the dosage of 104PFU in 0.2 ml, 3 weeks after vaccination, all chickens were challenged with 105ELD5o of F48E8 strain NDV by intranasal. Serum antibodies, morbidity and mortality were used to evaluate the efficacy of the rFPVs. Chickens vaccinated with rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVtp-SFB and inactivated vaccine in oil emulsion all survived, while the protection rate of the group inoculated with Wt-FPV was 0.
2.2 Protective efficacy of the rFPVs in commercial chickens
26-day-old commercial chickens were immunized with the four rFPVs respectively by SC route at the dosage of 105PFU, and chickens vaccinated with Wt-FPV, inactivated vaccine in oil emulsion and unvaccinated were used as control. 21 days after vaccination, all chickens were challenge with 105ELD50 of F48E8 strain NDV. The protection rates of rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVLp-SFB, and inactivated vaccine in oil emulsion were 64%, 60%, 52%, 88% and 100% respectively, while the chickens vaccinated with Wt-FPV and unvaccinated all died.
3 Development of indirect ELISA for detecting antibodies of fowlpox virus
The FPV propagated on CEF was harvested as antigen to develop an indirect ELISA for detecting antibodies of fowlpox virus. The optimal coating concentration of antigen was 2.7ug of FPV protein per well, the serum sample were diluted to 1:100 and the cutoff was determined to be 0.098. The indirect ELISA detected a total of 400 serum sample originated from chickens immunized with FPV or rFPV, and the positive efficacy was 81.25% (325/400). The test also revealed that indirect ELISA was 400-800 tunes more sensitive than AGP. Otherwise, it was simple, convenient, specific and rapid for detecting antibodies of fowlpox virus.
The findings showed that the four rFPVs we got not only had good genetic stability, but also provided adequate protection against the challenge of 105ELDso of F48E8 strain
NDV in SPF chicken. So the rFPVs vaccines will be a promising substitution of conventional vaccines to prevent and control NDV.
1 表达ZJ-1株NDV F基因重组鸡痘病毒的筛选及其遗传稳定性测定
利用FuGEN~(TM)6 Transfection Reagent进行转染，分别将含有NDV F基因的转移载体P11SF和PN11SF与282E4株和大空斑鸡痘病毒共转染鸡胚成纤维细胞(CEF)，通过蓝斑筛选，得到纯化的重组病毒rFPV_(282E4)-SF_A、rFPV_(LP)-SF_A、rFPV_(282E4)-SF_B和rFPV_(LP)-SF_B。PCR试验表明重组病毒基因组中含有F基因；间接免疫荧光试验(IFA)表明重组病毒中的F基因能够得到表达。
重组病毒经CEF连续传20代，覆盖含x-gal的琼脂，其在CEF上形成的空斑均为蓝斑。间接免疫荧光试验表明，重组病毒传20代后F基因的表达未受影响。取0、10和20代重组病毒DNA为模板进行PCR，均可扩增出约1.7 Kb的F基因片段。取rFPV_(282E4)-SF_B和rFPV_(LP)-SF_B的0、10和20代PCR产物进行测序后与转移载体中的F基因相比，核苷酸和氨基酸无任何变化，显示了该重组病毒具有良好的遗传稳定性。
2 重组病毒的动物试验
2．1 重组病毒对SPF鸡的免疫效力
5日龄SPF鸡随机分为6组，进行皮下接种W_1FPV_(LP)、rFPV_(282E4)-SF_A、
扬州大学硕士学位论文
rFPvL产SF^、亦PvZ:2以一SF。和:FPvLP一SFB lo4PFUz只，油苗为o.Zml/只，免疫后3
周龄用1护ELDS。F48E8株NDv进行滴鼻攻击。通过EusA抗体水平和保护效率
来评价重组病毒的免疫效力，结果除wtFPvLP组全部死亡外，其余组均100%保护。
2.2重组病毒对商品鸡的免疫效力
将26日龄商品鸡随机分为7组，以105PFu/只的量分别将wtFPvLP、
rFpVZ:2E4一sF^、rFpVLP一sFA、rFpVZsZE4一sF。和亦PVL产sF。进行皮下接种，油苗为
o.Zmlz只，另外设空白对照组。免疫后21天以lo5ELnso F4sEs株Nnv滴鼻攻毒，
观察14天，其保护效率rFPVZsZE4一SF^、rFPVLP一SF^、rFPVZsZE4一SFB和:FPvLP一SFs
分别为64%、60%、52%、88%;油苗和WtFPVLP分别为1 00%、0。
3应用间接ELIsA检测鸡痘病毒抗体方法的初步建立
应用CEF扩增的鸡痘病毒(FPV)提纯后作为抗原建立了具有较高特异性和
灵敏性的间接ELIsA方法来检测FPV抗体。通过方阵滴定法来确定抗原最佳包被
浓度为2.7u留孔，待检血清最佳稀释倍数为1:100，其阳性临界值为OD妻0.098。
将400份FPV或者重组鸡痘病毒免疫过的实验鸡血清用本方法进行检测，其阳性
检出率为81.25%(325/4 00)。此外，将该方法与琼脂扩散试验进行比较检测血清
样品，结果显示本方法的灵敏度比琼脂扩散试验灵敏400一800倍，而且还有特异性
强，操作简便、快速等优点。
综上所述，本试验成功筛选了4株重组鸡痘病毒，并且具有良好的遗传稳定
性，对SPF鸡能提供100%的保护。该疫苗的研制成功将会加速新型基因工程疫苗
替代传统灭活苗的步伐，并将为ND的防制带来新的技术手段。
Newcastle disease (ND) that caused by Newcastle disease virus (NDV) is one of the most important avian diseases. It is widespread in many country and areas throughout the world, and can cause severe economic losses in the poultry industry. NDV is a paramyxovirus possessing two major glycoprotein components of the virion envelope, one of which is the Fusion (F) protein. It is involved in membrane fusion and virus penetration, and is known to play an important role in immunity to the disease. In order to develop new genetic engineering vaccines which are safe and effective, we constructed four recombinant fowlpox virus (rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4 -SFB, rFPVLp-SFB) expressing F gene of ZJ-1 strain of NDV and evaluated its genetic stability and protective efficacy in this study.
1 Selection of recombinant fowlpox viruses (rFPVs) expressing F gene of NDV and their genetic stability
The transfer vectors P11SF and PN11SF were transfected by Fugend? Transfection Reagent mediated transfection on CEF infected with parent 282E4 and large plaque strain FPV. The four rFPVs ( rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVLp-SFB) were selected and purified by blue plaques expressing 3 -galactosidase. The insertion and expression of F genes in rFPVs were confirmed by PCR and IFA respectively.
Four rFPVs were serially passaged to the 20th on CEF monolayers, the expression of F genes were indentified by IFA. The F genes of passages 0, 10, and 20 were amplified by PCR and sequenced. There were no changes on the nucleotide and amino acid. The results showed that the four rFPVs were genetically stable for at least 20
passages.
2 Vaccination trials with rFPVs in chickens
2.1 Protective efficacy of the rFPVs in SPF chickens
Six groups of 5-day-old SPF chickens were vaccinated with Wt-FPVLp, rFPV282E4 -SFA, rFPVtp-SFA, rFPV282E4-SFB, rFPVtp-SFe and inactivated vaccine in oil emulsion by SC route in the nape of neck at the dosage of 104PFU in 0.2 ml, 3 weeks after vaccination, all chickens were challenged with 105ELD5o of F48E8 strain NDV by intranasal. Serum antibodies, morbidity and mortality were used to evaluate the efficacy of the rFPVs. Chickens vaccinated with rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVtp-SFB and inactivated vaccine in oil emulsion all survived, while the protection rate of the group inoculated with Wt-FPV was 0.
2.2 Protective efficacy of the rFPVs in commercial chickens
26-day-old commercial chickens were immunized with the four rFPVs respectively by SC route at the dosage of 105PFU, and chickens vaccinated with Wt-FPV, inactivated vaccine in oil emulsion and unvaccinated were used as control. 21 days after vaccination, all chickens were challenge with 105ELD50 of F48E8 strain NDV. The protection rates of rFPV282E4-SFA, rFPVLp-SFA, rFPV282E4-SFB, rFPVLp-SFB, and inactivated vaccine in oil emulsion were 64%, 60%, 52%, 88% and 100% respectively, while the chickens vaccinated with Wt-FPV and unvaccinated all died.
3 Development of indirect ELISA for detecting antibodies of fowlpox virus
The FPV propagated on CEF was harvested as antigen to develop an indirect ELISA for detecting antibodies of fowlpox virus. The optimal coating concentration of antigen was 2.7ug of FPV protein per well, the serum sample were diluted to 1:100 and the cutoff was determined to be 0.098. The indirect ELISA detected a total of 400 serum sample originated from chickens immunized with FPV or rFPV, and the positive efficacy was 81.25% (325/400). The test also revealed that indirect ELISA was 400-800 tunes more sensitive than AGP. Otherwise, it was simple, convenient, specific and rapid for detecting antibodies of fowlpox virus.
The findings showed that the four rFPVs we got not only had good genetic stability, but also provided adequate protection against the challenge of 105ELDso of F48E8 strain
NDV in SPF chicken. So the rFPVs vaccines will be a promising substitution of conventional vaccines to prevent and control NDV.
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45. Xu, Y., N. Francis, J. Szoka. 1996. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochem 35: 5616-5623.
46. Roif, M. 1997. Zinkeernagel Hans Hengartner Antiviral immunity. Immunology today 18(6): 258～259
47. Meulemans, G., C. Letelller, and M. Gonze, Et al. 1988. Newcastle disease virus F glycoprotein expressed from a recombinant vaccinia virus vector of protects chickens against live virus challenge. Avian Pathology 17: 821-827.
48. Wemers. C. D., S. Henau, C. Neyt, et al. 1987. The hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus strain Italian (ndv Italian): Comparison with HNs of other strains and expression by a vaccinia recombinant. Archives of Virology 97: 101-113.
49. Morgan, R. W., J. Gelb, C. S. Schreurs, et al. 1992. Protection of chickens from Newcastle and Mark's disease with a recombinant herpserivus of turkey's vaccine expressing the Newcastle disease virus fusion protein. Avian Disease 36: 858-870
50. Sakaguchi, M., H. Nakamura, k. Sonoda, et al. 1998. Protection of chickens with or without maternal antibodies against both Marek'S and Newcastle diseases by one -time vaccination with recombinant vaccine of Marek's disease virus type-1. Vaccine 16:
472-279
51．赵军，张秀根，陈溥言，等．表达鸡新城疫病毒HN基因的重组火鸡痘疹病毒的构建．畜牧兽医学报，2000，32(2)：150-154
52．张雪莲，魏荣，陈溥言，等．新城疫病毒F48E9病毒F基因表达盒的火鸡疱疹病毒转移质粒载体构建．中国兽医科技，2001，31(8)：5-7。
53. Lnae, J. M, F. Z. Ruben, J. M. Neff. 1969. Complications of smallpox vaccine 1968 National surveillance in the hired states New England. Journal of Medicine 281: 1201-1208
54. Boursnell, M. E., P. F. Green, J. I. Campbell, et al. 1990. Insertion of the fusion gene from Newcastle disease virus into a non—essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant. Journal of General Virology 71: 621—628.
55. Edbauer, C., R. weinberg, J. Taylor, et al. 1990. Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus hemagglutin in neuram inidase gene. Virology 179: 901—904
56. Taylor, J., C. J. Edbauer, R.J.Tayi, et al. 1990. Newcastle disease virus fusion protein expressed in a fowlopox virus recombinant confers protection in chickens. Gen virol 64: 1441-1450
57. Edbauar, C., R. Weinberg, R. J. Tayi, et al. 1990. Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus haema- glutinin-neurain in idase gene. Virology 79: 901-904
58. Ogawa, R., N. Yangida, E.Seakis, et al. 1990. Recombinant fowlpox viruses inducing protective immunity against Newcastle disease and fowlpox viruses. Vaccine 8: 486-490
59．曹殿军．刘惠莉，王莉林，等．新城疫病毒HN基因在重组鸡痘病毒中的表达及其保护性的研究．中国免疫学杂志．1998，14(1)：61-64
60．朱爱华．彭大新，等．痘病毒载体强启动子的构建和筛选．扬州大学学报．1999(2)：25-28
61．金宁一．殷震．等．鸡痘病毒282E4弱毒株TK基因限制性酶切图谱分析．中国兽医学报．1994，14(1)：5-13。
62．郭志儒．金宁一，王兴龙，等．鸡痘病毒毒282E4株表达载体的构建及新城疫病毒F蛋白的表达．中国兽医学报，2000．20(5)：9423-428
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43．陈吉祥，薛飞群．CTAB脂质体介导鸡新城疫病毒核酸免疫研究．动物医学进展，2001，22(1)：41-43
44. Pinnadumage, P., L. Schmitt, L. Hang. 1987. Use of a quaternary anmonium detergent in liposome mediated DNA transfection of mouse L-cells. Bio chin Biohys Acta 985: 33-37
45. Xu, Y., N. Francis, J. Szoka. 1996. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochem 35: 5616-5623.
46. Roif, M. 1997. Zinkeernagel Hans Hengartner Antiviral immunity. Immunology today 18(6): 258～259
47. Meulemans, G., C. Letelller, and M. Gonze, Et al. 1988. Newcastle disease virus F glycoprotein expressed from a recombinant vaccinia virus vector of protects chickens against live virus challenge. Avian Pathology 17: 821-827.
48. Wemers. C. D., S. Henau, C. Neyt, et al. 1987. The hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus strain Italian (ndv Italian): Comparison with HNs of other strains and expression by a vaccinia recombinant. Archives of Virology 97: 101-113.
49. Morgan, R. W., J. Gelb, C. S. Schreurs, et al. 1992. Protection of chickens from Newcastle and Mark's disease with a recombinant herpserivus of turkey's vaccine expressing the Newcastle disease virus fusion protein. Avian Disease 36: 858-870
50. Sakaguchi, M., H. Nakamura, k. Sonoda, et al. 1998. Protection of chickens with or without maternal antibodies against both Marek'S and Newcastle diseases by one -time vaccination with recombinant vaccine of Marek's disease virus type-1. Vaccine 16:
472-279
51．赵军，张秀根，陈溥言，等．表达鸡新城疫病毒HN基因的重组火鸡痘疹病毒的构建．畜牧兽医学报，2000，32(2)：150-154
52．张雪莲，魏荣，陈溥言，等．新城疫病毒F48E9病毒F基因表达盒的火鸡疱疹病毒转移质粒载体构建．中国兽医科技，2001，31(8)：5-7。
53. Lnae, J. M, F. Z. Ruben, J. M. Neff. 1969. Complications of smallpox vaccine 1968 National surveillance in the hired states New England. Journal of Medicine 281: 1201-1208
54. Boursnell, M. E., P. F. Green, J. I. Campbell, et al. 1990. Insertion of the fusion gene from Newcastle disease virus into a non—essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant. Journal of General Virology 71: 621—628.
55. Edbauer, C., R. weinberg, J. Taylor, et al. 1990. Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus hemagglutin in neuram inidase gene. Virology 179: 901—904
56. Taylor, J., C. J. Edbauer, R.J.Tayi, et al. 1990. Newcastle disease virus fusion protein expressed in a fowlopox virus recombinant confers protection in chickens. Gen virol 64: 1441-1450
57. Edbauar, C., R. Weinberg, R. J. Tayi, et al. 1990. Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus haema- glutinin-neurain in idase gene. Virology 79: 901-904
58. Ogawa, R., N. Yangida, E.Seakis, et al. 1990. Recombinant fowlpox viruses inducing protective immunity against Newcastle disease and fowlpox viruses. Vaccine 8: 486-490
59．曹殿军．刘惠莉，王莉林，等．新城疫病毒HN基因在重组鸡痘病毒中的表达及其保护性的研究．中国免疫学杂志．1998，14(1)：61-64
60．朱爱华．彭大新，等．痘病毒载体强启动子的构建和筛选．扬州大学学报．1999(2)：25-28
61．金宁一．殷震．等．鸡痘病毒282E4弱毒株TK基因限制性酶切图谱分析．中国兽医学报．1994，14(1)：5-13。
62．郭志儒．金宁一，王兴龙，等．鸡痘病毒毒282E4株表达载体的构建及新城疫病毒F蛋白的表达．中国兽医学报，2000．20(5)：9423-428