鸡球虫3-1E抗原和NA4抗原对4种艾美耳球虫交叉免疫保护作用的研究
摘要
鸡球虫病(Coccidiosis)是由艾美耳球虫(Eimeria)引起的一种严重危害养禽业发展的寄生原虫病,传统的防制办法还存在一些问题。核酸疫苗是一种新的生物技术疫苗,具有可靠的安全性、高稳定性、能诱导机体产生全面的免疫应答等优点。近年来,鸡球虫核酸疫苗的研究取得了重要进展。
本实验室已从E.acervulina和E.necatrix孢子化卵囊分别克隆得到免疫保护性抗原基因3-1E和NA4,并利用DNA重组技术成功构建了免疫调节型DNA疫苗pVAX1.0-NA4-IL-2和pVAX1.0-3-1E-IL-2,实验表明这两种DNA疫苗对本种球虫感染均具有较好的免疫保护作用。
用DNAStar中的MegAlign软件对3-1E和NA4抗原的基因进行分析,结果发现3-1E抗原在E.tenella和E.acervulina之间基因序列的同源性达99.8%,氨基酸序列的同源性为99.4%;NA4抗原与E.tenella的TA4抗原之间基因序列的同源性达91.4%,氨基酸序列的同源性为86%。用Protean软件对3-1E和NA4抗原的二级结构和抗原性进行预测分析,结果表明这两个抗原均具有很好的抗原性。
将免疫调节型DNA疫苗pVAX1.0-3-1E-IL-2经腿部肌肉分别于14、21日龄两次免疫雏鸡,28日龄经口接种新鲜的E.tenella、E.necatrix、E.acervulina和E.maxima孢子化卵囊,34日龄宰杀全部实验动物,分别作肠道病变计分、肠道卵囊计数、增重和存活率几个指标测定,以抗球虫指数(ACI)综合判定其保护性。结果表明,免疫调节型DNA疫苗pVAX1.0-3-1E-IL-2抗E.tenella、E.necatrix、E.acervulina和E.maxhna的ACI值分别为180.11、174.24、180.64和164.74。
用同样的方法对免疫调节型DNA疫苗pVAX1.0-NA4-IL-2进行交叉免疫保护性实验,结果发现,pVAX1.0-NA4-IL-2抗E.tenella、E.necatrix、E.acervulina和E.maxima的ACI分别为180.35、181.20、171.63和161.89。
本研究通过序列分析和动物交叉免疫保护性实验分别证明了鸡球虫3-1E抗原和NA4抗原对四种艾美耳球虫均具有部分交叉免疫保护作用,这两种抗原有可能作为艾美耳球虫交叉免疫保护性DNA疫苗的候选抗原。
The coccidiosis of chickens is a protozoan parasite disease caused by Eimeria,which jeopardizes severely the development of the poultry industry.However,there exists some problems in traditional protection and controls.Nucleic acid vaccine is a new type of bio-technological vaccine characterized by credible security,high stability,and the ability that can induce immune response all over the body.In recent years,great advances have been made on the research of nucleic acid vaccine against chicken coccidiosis.
Genes of protective-immune antigens 3-1E and NA4 had been respectively cloned from sporulated oocysts of E.acervulina and E.necatrix at the laboratory of parasitology at NAU,and immune-regulating DNA vaccines of pVAX1.0-NA4-IL-2 and pVAX1.0-3-1E-IL-2 had been constructed successfully by DNA recombinant assay.The test results indicated that the two type of vaccines had significant protective-immune effects against infection with E.necatrix and E.tenella.
Genes of antigens 3-1E and NA4 were analyzed by the sofeware of MegAlign from DNAStar.It turned out to have been discovered that the gene sequence homology of antigen 3-1E between E.tenella and E.acervulina was up to 99.8%,and amino acid sequence homology of that reached 99.4%.While the homologous gene sequences and the homologous amino acid sequences between antigen NA4 and antigen TA4 against E.tenella were 91.4%and 86%respectively.The secondary structure and the antigeniciy of antigen 3-1E and antigen NA4 were disposed by forecasting analysis through the sofeware of Protean,and the results showed that both of the two antigens were outstanding in antigenicity.
Chickens were vaccinated through leg by the immune-regulating vaccine of pVAX1.0-3-1E-IL-2 once at age of 14 days and 21 days,then were inoculated with sporulated oocysts of E.tenella、E.necatrix、E.acervulina and E.maxima respectively at age of 28 days.All experimental animals were killed at age of 34 days,and indexs including lesion scores of intestine,the number of intestinal oocysts,weight gain,and livability were determined for comprehensive estimate on the protection by anti-coccidial index(ACI).The results revealed that ACI values of the immune-regulating DNA vaccine pVAX1.0-3-1E-IL-2 against E.tenella,E.necatrix,E.acervulina,and E.maxima were 180.11, 174.24,180.64,and 164.74 respectively.
By the same approach mentioned above,the immune-regulating DNA vaccine pVAX1.0-NA4-IL-2 was tested by the trial of cross-protective immunity.It was found that ACI values of pVAX1.0-NA4-IL-2 against E.tenella,E.necatrix,E.acervulina,and E.maxima were 180.35,181.20,171.63,and 161.89 respectively.
In the present study,it was proved by sequence analysis and animal test of cross-protective immunity that antigen 3-1E and antigen NA4 against chickens had cross-protective immunity against four species of Eimeria.And the two varieties of antigens had the potential to become antigen candidates as cross-protective immune DNA vaccines against Eimeria.
本实验室已从E.acervulina和E.necatrix孢子化卵囊分别克隆得到免疫保护性抗原基因3-1E和NA4,并利用DNA重组技术成功构建了免疫调节型DNA疫苗pVAX1.0-NA4-IL-2和pVAX1.0-3-1E-IL-2,实验表明这两种DNA疫苗对本种球虫感染均具有较好的免疫保护作用。
用DNAStar中的MegAlign软件对3-1E和NA4抗原的基因进行分析,结果发现3-1E抗原在E.tenella和E.acervulina之间基因序列的同源性达99.8%,氨基酸序列的同源性为99.4%;NA4抗原与E.tenella的TA4抗原之间基因序列的同源性达91.4%,氨基酸序列的同源性为86%。用Protean软件对3-1E和NA4抗原的二级结构和抗原性进行预测分析,结果表明这两个抗原均具有很好的抗原性。
将免疫调节型DNA疫苗pVAX1.0-3-1E-IL-2经腿部肌肉分别于14、21日龄两次免疫雏鸡,28日龄经口接种新鲜的E.tenella、E.necatrix、E.acervulina和E.maxima孢子化卵囊,34日龄宰杀全部实验动物,分别作肠道病变计分、肠道卵囊计数、增重和存活率几个指标测定,以抗球虫指数(ACI)综合判定其保护性。结果表明,免疫调节型DNA疫苗pVAX1.0-3-1E-IL-2抗E.tenella、E.necatrix、E.acervulina和E.maxhna的ACI值分别为180.11、174.24、180.64和164.74。
用同样的方法对免疫调节型DNA疫苗pVAX1.0-NA4-IL-2进行交叉免疫保护性实验,结果发现,pVAX1.0-NA4-IL-2抗E.tenella、E.necatrix、E.acervulina和E.maxima的ACI分别为180.35、181.20、171.63和161.89。
本研究通过序列分析和动物交叉免疫保护性实验分别证明了鸡球虫3-1E抗原和NA4抗原对四种艾美耳球虫均具有部分交叉免疫保护作用,这两种抗原有可能作为艾美耳球虫交叉免疫保护性DNA疫苗的候选抗原。
The coccidiosis of chickens is a protozoan parasite disease caused by Eimeria,which jeopardizes severely the development of the poultry industry.However,there exists some problems in traditional protection and controls.Nucleic acid vaccine is a new type of bio-technological vaccine characterized by credible security,high stability,and the ability that can induce immune response all over the body.In recent years,great advances have been made on the research of nucleic acid vaccine against chicken coccidiosis.
Genes of protective-immune antigens 3-1E and NA4 had been respectively cloned from sporulated oocysts of E.acervulina and E.necatrix at the laboratory of parasitology at NAU,and immune-regulating DNA vaccines of pVAX1.0-NA4-IL-2 and pVAX1.0-3-1E-IL-2 had been constructed successfully by DNA recombinant assay.The test results indicated that the two type of vaccines had significant protective-immune effects against infection with E.necatrix and E.tenella.
Genes of antigens 3-1E and NA4 were analyzed by the sofeware of MegAlign from DNAStar.It turned out to have been discovered that the gene sequence homology of antigen 3-1E between E.tenella and E.acervulina was up to 99.8%,and amino acid sequence homology of that reached 99.4%.While the homologous gene sequences and the homologous amino acid sequences between antigen NA4 and antigen TA4 against E.tenella were 91.4%and 86%respectively.The secondary structure and the antigeniciy of antigen 3-1E and antigen NA4 were disposed by forecasting analysis through the sofeware of Protean,and the results showed that both of the two antigens were outstanding in antigenicity.
Chickens were vaccinated through leg by the immune-regulating vaccine of pVAX1.0-3-1E-IL-2 once at age of 14 days and 21 days,then were inoculated with sporulated oocysts of E.tenella、E.necatrix、E.acervulina and E.maxima respectively at age of 28 days.All experimental animals were killed at age of 34 days,and indexs including lesion scores of intestine,the number of intestinal oocysts,weight gain,and livability were determined for comprehensive estimate on the protection by anti-coccidial index(ACI).The results revealed that ACI values of the immune-regulating DNA vaccine pVAX1.0-3-1E-IL-2 against E.tenella,E.necatrix,E.acervulina,and E.maxima were 180.11, 174.24,180.64,and 164.74 respectively.
By the same approach mentioned above,the immune-regulating DNA vaccine pVAX1.0-NA4-IL-2 was tested by the trial of cross-protective immunity.It was found that ACI values of pVAX1.0-NA4-IL-2 against E.tenella,E.necatrix,E.acervulina,and E.maxima were 180.35,181.20,171.63,and 161.89 respectively.
In the present study,it was proved by sequence analysis and animal test of cross-protective immunity that antigen 3-1E and antigen NA4 against chickens had cross-protective immunity against four species of Eimeria.And the two varieties of antigens had the potential to become antigen candidates as cross-protective immune DNA vaccines against Eimeria.
引文
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[1]索勋,李国清主编.鸡球虫病学[M].北京:中国农业大学出版社,1998.
[2]高福,刘文军主译.禽病学[M].第9版.北京:北京农业大学出版社,1991.682-695.
[3]Karel Z Mewman,John L Tedesco,Thomas CGore,Gary Petersen,Virginia M Brothers,James G Files,Leland S Paul assigned to Solvay & Cie S A 5028694 Antigenic proteins and vaccines containing them for prevention of coccidiosis caused by eimeria eimeria necatrix and eimeria tenella[J].Biotechnology Advances,1991,9(3):539.
[4]J萨姆布鲁克,D.W.拉塞尔著,黄培堂等译,分子克隆实验指南[M].(第三版).科学出版社.2002.8:522-525.
[5]Johnson J,Reid W M.Anticoccidial drugs:lesion scoring techniques in battery and floorpen experiments with chickens[J].Exp Parasitol,1970,28:30-36.
[6]潘晓亮,丁熙成,蒋金书.TA4和Et-MIC2表达产物免疫后对感染柔嫩艾美耳球虫(Eimeria tenella)鸡增重和盲肠卵囊数的影响[J].黑龙江畜牧兽医,2002,8:6-7.
[7]胡子信,门常平.病毒核酸疫苗研究进展[J].1997,中国兽医学报,12(2):83-88.