猪瘟病毒E2基因真核表达质粒的构建及基因疫苗的研究
作者：陈创夫 余兴龙 涂长春 李红卫 李作生 马正海 孙明 殷震
单位：陈创夫(新疆塔里木农垦大学，新疆阿拉尔 843300)；余兴龙 涂长春 李红卫 李作生 马正海 孙明 殷震(解放军农牧大学 军事兽医研究所，长春 130062)
关键词：猪瘟病毒；E2基因；真核表达质粒；基因疫苗
中国病毒学000310摘要：构建了猪瘟病毒(classical swine fever virus, CSFV)主要保护性抗原E2基因4种不同的真核表达质粒。小鼠免疫试验表明，E2基因上不同的功能区对基因疫苗的免疫应答有很大影响，有信号肽序列的E2基因可诱导产生特异性免疫反应，且无跨膜区序列的E2基因所诱导的免疫应答反应比有跨膜区序列的强，而无信号肽序列的E2基因则不能诱导产生CSFV特异性的免疫反应。攻毒保护试验表明，免疫家兔最少可抵抗10个最小感染剂量(MID)的猪瘟兔化弱毒苗(Hog cholera lap-inized virus, HCLV)的攻击；免疫猪可抵抗致死剂量的CSFV石门株强毒的攻击。
, http://www.100md.com
中图分类号：Q78，S852.4 文献标识码：A
文章编号：1003-5125(2000)03-0264-08
Construction of Eukaryotic Expression Plasmids of
CSFV E2 Gene and the Study on DNA Vaccine
YU Xing-long，TU Chang-chun，LI Hong-wei，CHEN Chuang-fu，LI Zuo-sheng，MA Zheng-hai，YIN Zhen
(Changchun University of Agricultural and Animal Science, Changchun, 130062, China)
, 百拇医药
Abstract: Four kinds of eukaryotic expressing plasmids of classical swine fever virus (CSFV) E2 gene, either containing or lacking the signal sequence and transmembrane region sequence, were constructed. The antibodies against CSFV were induced after immunization of mice by intramuscular (IM) injection with the plasmids of E2 gene with a signal sequence, pcDST and pcDSW, but not with the plasmids of E2 gene lacking a signal sequence, pcDWT and pcDWW. The level of antibodies elicited by pcDSW lacking transmembrane region sequence was higher than that elicited by pcDST with transmembrane region sequence. The results indicated the plasmid expressing a secreted antigen may be more efficient for induction of immune response when administered by IM injection than that expressing non-secreted antigen. Immunization with pcDSW could protect rabbits against challenge with at least 10 MID hog cholera lapinized virus (HCLV) and protect pig from the challenge of lethal dose of CSFV strain, Shimenmen.
, 百拇医药
Key words: CSFV; E2 gene; Eukaryotic expression; Plasmid; DNA vaccine
基金项目：国家九五科技攻关计划资助项目(96C010401)
作者简介：余兴龙(1965年-)，男，湖南省平江县人，助理研究员，博士，主要从事
动物病毒的防治研究工作。
参考文献
[1] Wolff J A, Malone R, Williams P, et al. Direct gene transfer into mouse muscle in vivo [J]. Science, 1990,247:1465-1468
[2] Tang D, Devit M, Johnston S A. Genetic immunization is a simple method for eliciting an immune response [J]. Nature, 1992,356:152-154
, 百拇医药
[3] 谢庆阁，翟中和主编.畜禽重大疫病免疫防制研究[M].北京：中国农业科技出版社，1997，22-26
[4] Wichs I P, Howell M L, Hancock T, et al. Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy [J]. Hum Gene Ther, 1995,6:317-323
[5] 苏慧慈，刘彦仿著.原位PCR[M].北京：科学出版社.1997.39-81
[6] 余兴龙，涂长春，李作生，等.以重组mE2蛋白为抗原建立检测猪瘟病毒抗体间接ELISA方法的研究[J].中国预防兽医学报，1999，21(3)：220-222
, http://www.100md.com
[7] Kozak M. The scanning model for translation: an update [J]. J Cell Biol, 1989,108:229-241
[8] Kozak M. At least six nucleotide preceding the AUG initiator codon enhance translation in mammalian cells [J]. J Mol Biol. 1987,196:947-950
[9] Rumenape T, Unger G, Strauss J H, et al. Processing of the envelope glycoproteins of pestiviruses [J]. J Virol, 1993,3288-3294
[10] Haddad D, Liljeqvist S, Stahl S, et al. Comparative study of DNA-based immunizationvectors: effect of secretion signals on the antibody response [J]. J Virol, 1998,72:1497-1503
, http://www.100md.com
[11] Zijl M, Wensvoort G, Kluyver E, et al. Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholear virus protects swine against both pseudorabies and hog cholera [J]. J Virol, 1991,65:2761-2765
[12] Hariharan M J, Driver D A, Townsend K, et al. DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector [J]. J Virol, 1998,72:950-958
[13] 余兴龙，涂长春，李作生，等.猪IL-2和IL-6 cDNA的克隆及序列测定[J].中国兽医学报，1999，19：39
收稿日期：1999-03-11，修回日期：1999-09-10, 百拇医药
单位：陈创夫(新疆塔里木农垦大学，新疆阿拉尔 843300)；余兴龙 涂长春 李红卫 李作生 马正海 孙明 殷震(解放军农牧大学 军事兽医研究所，长春 130062)
关键词：猪瘟病毒；E2基因；真核表达质粒；基因疫苗
中国病毒学000310摘要：构建了猪瘟病毒(classical swine fever virus, CSFV)主要保护性抗原E2基因4种不同的真核表达质粒。小鼠免疫试验表明，E2基因上不同的功能区对基因疫苗的免疫应答有很大影响，有信号肽序列的E2基因可诱导产生特异性免疫反应，且无跨膜区序列的E2基因所诱导的免疫应答反应比有跨膜区序列的强，而无信号肽序列的E2基因则不能诱导产生CSFV特异性的免疫反应。攻毒保护试验表明，免疫家兔最少可抵抗10个最小感染剂量(MID)的猪瘟兔化弱毒苗(Hog cholera lap-inized virus, HCLV)的攻击；免疫猪可抵抗致死剂量的CSFV石门株强毒的攻击。
, http://www.100md.com
中图分类号：Q78，S852.4 文献标识码：A
文章编号：1003-5125(2000)03-0264-08
Construction of Eukaryotic Expression Plasmids of
CSFV E2 Gene and the Study on DNA Vaccine
YU Xing-long，TU Chang-chun，LI Hong-wei，CHEN Chuang-fu，LI Zuo-sheng，MA Zheng-hai，YIN Zhen
(Changchun University of Agricultural and Animal Science, Changchun, 130062, China)
, 百拇医药
Abstract: Four kinds of eukaryotic expressing plasmids of classical swine fever virus (CSFV) E2 gene, either containing or lacking the signal sequence and transmembrane region sequence, were constructed. The antibodies against CSFV were induced after immunization of mice by intramuscular (IM) injection with the plasmids of E2 gene with a signal sequence, pcDST and pcDSW, but not with the plasmids of E2 gene lacking a signal sequence, pcDWT and pcDWW. The level of antibodies elicited by pcDSW lacking transmembrane region sequence was higher than that elicited by pcDST with transmembrane region sequence. The results indicated the plasmid expressing a secreted antigen may be more efficient for induction of immune response when administered by IM injection than that expressing non-secreted antigen. Immunization with pcDSW could protect rabbits against challenge with at least 10 MID hog cholera lapinized virus (HCLV) and protect pig from the challenge of lethal dose of CSFV strain, Shimenmen.
, 百拇医药
Key words: CSFV; E2 gene; Eukaryotic expression; Plasmid; DNA vaccine
基金项目：国家九五科技攻关计划资助项目(96C010401)
作者简介：余兴龙(1965年-)，男，湖南省平江县人，助理研究员，博士，主要从事
动物病毒的防治研究工作。
参考文献
[1] Wolff J A, Malone R, Williams P, et al. Direct gene transfer into mouse muscle in vivo [J]. Science, 1990,247:1465-1468
[2] Tang D, Devit M, Johnston S A. Genetic immunization is a simple method for eliciting an immune response [J]. Nature, 1992,356:152-154
, 百拇医药
[3] 谢庆阁，翟中和主编.畜禽重大疫病免疫防制研究[M].北京：中国农业科技出版社，1997，22-26
[4] Wichs I P, Howell M L, Hancock T, et al. Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy [J]. Hum Gene Ther, 1995,6:317-323
[5] 苏慧慈，刘彦仿著.原位PCR[M].北京：科学出版社.1997.39-81
[6] 余兴龙，涂长春，李作生，等.以重组mE2蛋白为抗原建立检测猪瘟病毒抗体间接ELISA方法的研究[J].中国预防兽医学报，1999，21(3)：220-222
, http://www.100md.com
[7] Kozak M. The scanning model for translation: an update [J]. J Cell Biol, 1989,108:229-241
[8] Kozak M. At least six nucleotide preceding the AUG initiator codon enhance translation in mammalian cells [J]. J Mol Biol. 1987,196:947-950
[9] Rumenape T, Unger G, Strauss J H, et al. Processing of the envelope glycoproteins of pestiviruses [J]. J Virol, 1993,3288-3294
[10] Haddad D, Liljeqvist S, Stahl S, et al. Comparative study of DNA-based immunizationvectors: effect of secretion signals on the antibody response [J]. J Virol, 1998,72:1497-1503
, http://www.100md.com
[11] Zijl M, Wensvoort G, Kluyver E, et al. Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholear virus protects swine against both pseudorabies and hog cholera [J]. J Virol, 1991,65:2761-2765
[12] Hariharan M J, Driver D A, Townsend K, et al. DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector [J]. J Virol, 1998,72:950-958
[13] 余兴龙，涂长春，李作生，等.猪IL-2和IL-6 cDNA的克隆及序列测定[J].中国兽医学报，1999，19：39
收稿日期：1999-03-11，修回日期：1999-09-10, 百拇医药