【作者】 宋小凯；

【导师】 李祥瑞；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2008， 博士

【摘要】 对柔嫩艾美耳球虫免疫调节型DNA疫苗pcDNA3.1-TA4-IL-2的免疫剂量进行筛选。设计了200μg、100μg、50μg、25μg四个剂量组以及非免疫攻虫和非免疫非攻虫两个对照组。按照相应的免疫剂量,实验组14日龄腿部肌肉注射DNA疫苗pcDNA-TA4-IL-2,21日龄加强免疫,对照组注射TE。28日龄,除非免疫非攻虫组外,其余均经口接种新鲜的柔嫩艾美耳球虫孢子化卵囊5×10~4个/羽。7天后剖杀所有实验鸡,检测其免疫保护效果。结果发现,25μg和50μg组的ACI分别为196.45和182.67,免疫保护效果明显;100μg组的ACI为172.16,免疫保护效果一般;200μg组的ACI为157,无免疫保护效果。对柔嫩艾美耳球虫免疫调节型DNA疫苗pcDNA3.1-TA4-IL-2的免疫途径进行筛选。设计了皮下注射、口服、静脉注射、肌肉注射、滴鼻滴眼五个实验组以及非免疫攻虫和非免疫非攻虫两个对照组。14日龄,实验组每只鸡按相对应的途径免疫接种25μg DNA疫苗,21日龄加强免疫,对照组注射TE。28日龄,除非免疫非攻虫组外,其余均经口接种新鲜的柔嫩艾美耳球虫孢子化卵囊5×10~4个/羽。7天后剖杀所有实验鸡,检测其免疫保护效果。结果发现,所有实验组的ACI都大于160。静脉注射组、皮下注射组和滴鼻、滴眼组的ACI分别为173.38、171.44、168.17,免疫保护效果一般。口服组的ACI为180.17,免疫保护效果明显。腿部肌肉注射组ACI最高,达到193.97,免疫保护效果最好。对DNA疫苗pcDNA3.1-TA4-IL-2的首免日龄和免疫次数进行筛选。设计了一周龄首免不加强免疫、一周龄首免二周龄加强免疫、二周龄首免不加强免疫、二周龄首免三周龄加强免疫四个实验组以及非免疫攻虫和非免疫非攻虫两个对照组。按照相应的免疫日龄与次数,实验组每只鸡经腿部肌肉注射25μg DNA疫苗,对照组注射TE。28日龄,除非免疫非攻虫组外,其余均经口接种新鲜的柔嫩艾美耳球虫孢子化卵囊5×10~4个/羽。7天后剖杀所有实验鸡,检测其免疫保护效果。结果发现,所有实验组的ACI都大于160,其中一周龄首免组比二周龄首免效果好;加强免疫组比非加强组效果差别不大;一周龄首免,二周龄加强免疫组效果最好。对DNA疫苗pcDNA3.1-TA4-IL-2的稳定性进行了研究。DNA疫苗在不同的温度(即-20℃、4℃和室温)下保存分别保存了1个月、3个月、6个月。分别用这些在不同条件保存过的DNA疫苗腿部肌肉注射免疫7日龄鸡,14日龄加强免疫。对照组注射TE。28日龄,除非免疫非攻虫组外,其余均经口接种新鲜的柔嫩艾美耳球虫孢子化卵囊5×10~4个/羽。7天后剖杀所有实验鸡,检测其免疫保护效果。结果发现,所有实验组的ACI都大于160,说明在本实验中所有DNA疫苗都有免疫保护效果。随着保存期的延长,疫苗的免疫保护效果稍有下降。随着保存温度的不同,疫苗的保护效果稍有变化。说明DNA疫苗在6个月内受温度和保存期影响小。对DNA疫苗pcDNA3.1-TA4-IL-2的交叉免疫保护行进行了研究。7日龄雏鸡经腿部肌肉注射DNA疫苗DNA25μg/羽,14日龄加强免疫.对照组注射TE。28日龄,除非免疫非攻虫组外,其余分别经口接种新鲜的柔嫩艾美耳球虫、毒害艾美耳球虫、堆型艾美耳球虫和巨型艾美耳球虫孢子化卵囊5×10~4个/羽。7天后剖杀所有实验鸡,检测其免疫保护效果。结果发现,柔嫩艾美耳球虫DNA疫苗pcDNA3.1-TA4-IL-2对毒害艾美耳球虫和堆型艾美耳球虫感染有部分交叉免疫保护作用,对巨型艾美耳球虫感染没有交叉免疫保护作用。利用DNAstar软件对柔嫩艾美耳球虫子孢子阶段抗原基因SO7和第二代裂殖子阶段抗原基因MZ5-7进行表位分析,选定T细胞表位比较集中的片断。结果显示,ml为MZ5-7基因的第115位到第435位的321个核苷酸,编码107个氨基酸;m2为MZS-7基因的第547位到第897位的351个核苷酸,编码117个氨基酸;s1为SO7基因的第7位到第291位的285个核苷酸,编码95个氨基酸;s2为SO7基因的第370位到第609位的240个核苷酸,编码80个氨基酸。根据所构建的重组质粒对酶切位点以及对阅读框的不同要求,利用软件primer premier5.0设计若干对特异性引物,分别以pMD18-T-SO7和pMD18-T-MZ5-7为模板,PCR扩增得到m1、m2、s1和s2,分别连入pMD18-T载体。鉴定结果显示,各个片断成功克隆到pMD18-T载体中。把s1、s2、m1和m2,以不同的组合克隆到pVAX1载体中,构建了多价表位DNA疫苗:pVAX1-m1-m2-s1-s2、pVAX1-m1-s1、pVAX1-m1-s2、pVAX1-m2-s1、pVAX1-m2-s2;表位DNA疫苗:pVAX1-m1、pVAX1-m2、pVAX1-s1和pVAX1-s2。分别连接chIFN-γ和chIL-2作为基因佐剂构建了免疫调节型多价表位DNA疫苗:pVAX1-m1-m2-s1-s2-IFN□、pVAX1-m1-m2-s1-s2-IL2、pVAX1-m1-s1-IFN□、pVAX1-m1-s1-IL2、pVAX1-m1-s2-IFN□、pVAX1-m1-s2-IL2、pVAX1-m2-s1-IFN□、pVAX1-m2-s1-IL2、pVAX1-m2-s2-IFN□和pVAX1-m2-s2-IL2。酶切鉴定正确后,分别用重组质粒免疫7日龄鸡,1周后取注射部位、非注射部位肌肉组织,用RT-PCR,Western-blot检测保护性抗原的表达情况。结果表明,目的基因片段均能在注射部位肌肉里成功表达。对已构建好的19种重组质粒进行动物免疫保护性实验.设计了10个免疫调节型多价表位DNA疫苗组:pVAX1-m1-m2-s1-s2-IFN□、pVAX1-m1-m2-s1-s2-IL2、pVAX1-m1-s1-IFN□、pVAX1-m1-s1-IL2、pVAX1-m1-s2-IFN□、pVAX1-m1-s2-IL2、pVAX1-m2-s1-IFN□、pVAX1-m2-s1-IL2、pVAX1-m2-s2-IFN□、pVAX1-m2-s2-IL2;5个多价表位DNA疫苗组:pVAX1-m1-m2-s1-s2、pVAX1-m1-s1、pvAX1-m1-s2、pVAX1-m2-s1、pVAX1-m2-s2;4个表位DNA疫苗组:pVAX1-m1、pVAX1-m2、pVAX1-s1、pVAX1-s2;3个对照组:pVAX1空载体、非免疫非攻虫和非免疫攻虫对照组。用这19种重组质粒经腿部肌肉分别于14、21日龄两次免疫雏鸡(100μg/只),空载体对照组注射pVAX1,另两个对照组注射TE。28日龄,除非免疫非攻虫组外,其余鸡均经口接种新鲜的柔嫩艾美耳球虫孢子化卵囊5×10~4个/羽。一周后剖杀所有实验鸡,检测其免疫保护效果。结果表明,所构建的免疫调节型DNA疫苗对感染球虫鸡具有良好的保护效果。多价表位DNA疫苗诱导的免疫保护效果比单一阶段的DNA疫苗诱导免疫保护效果好,并且含有的表位越多,其免疫保护效果也越好。串联有细胞因子的免疫调节型多表位DNA疫苗的免疫保护效果比没有串连细胞因子的多表位DNA疫苗的免疫保护效果好,连接IFN□的DNA疫苗的免疫保护效果比连接IL-2的DNA疫苗的免疫保护效果好。其中以pVAX1-m1-m2-s1-s2-IFN□的免疫保护效果最好。更多还原

【Abstract】 The immunization dose of immune-regulative DNA vaccine pcDNA3.1-TA4-IL-2 was optimized.Four experimental groups i.e.200μg,100μg,50μg,and 25μg dose groups were designed and the challenged and unchallenged control groups were also designed.At the age of 14 days,experimental chickens were intramuscularly immunized with the corresponding immunization doses.A booster immunization was given by the same method as the first immunization 7 days later.The control groups were intramuscularly immunized with TE.At 28 days of age,the chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered.Body-weight gain,oocysts per gramfeces(OPG),lesion score, and ACI of each group were calculated.The results illustrated that ACIs of groups 25μg and 50μg were 196.45,182.67,respectively,which showed significantly effective protection against avian coccidiosis;ACI of group 100μg was 172.16,which showed effective protection against avian coccidiosis;ACI of group 200μg was 157,which showed ineffective protection against avian coccidiosis.The immunization route of DNA vaccine pcDNA3.1-TA4-IL-2 was optimized.Five experimental groups i.e.subcutaneous injection,oral administration,intravenous injection, intramuscular injection and intranasal administration groups were designed and the challenged and unchallenged control groups were also designed.At the age of 14 days, every experimental chicken was immunized with 25μg DNA vaccine by the corresponding immunization route.A booster immunization was given by the same method as the first immunization 7 days later.The control groups were immunized with TE.At 28 days of age, chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered. Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that ACIs of experimental groups were beyond 160, which showed effective protection against avian coccidiosis.ACIs of intravenous injection, subcutaneous injection and intranasal administration groups were 173.38,171.44,168.17, respectively,which showed effective protection against avian coccidiosis.ACI of oral administration was 180.04,which showed significantly effective protection against avian coccidiosis.Intramuscular injection induced a highest ACI of 193.97,which demonstrated intramuscular injection was the best immunization route of the five immunization routes.The primary immunization age and immunization times of DNA vaccine pcDNA3.1-TA4-IL-2 was optimized.Four experimental groups i.e.group immunized once at 7 days old,group immunized twice at 7 and 14 days old respectively,group immunized once at 14 days old,and group immunized twice at 14 and 21 days old respectively were designed and the challenged and unchallenged control groups were also designed. According to the corresponding primary immunization age and immunization times,every experimental chicken was intramuscularly immunized with 25μg DNA vaccine.The control groups were immunized with TE.At 28 days of age,chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered.Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that ACIs of experimental groups were beyond 160,which showed effective protection against avian coccidiosis.Younger primary immunization age groups showed a little higher ACI than older primary immunization age groups.There was no significant difference between the booster immunization groups and non-booster immunization groups.The stability of DNA vaccine pcDNA3.1-TA4-IL-2 was analysised.The DNA vaccines were stored at -20℃,4℃and room temperature for 1 month,3 months,and 6 months,respectively.With 25μg of the stored DNA vaccines,7-day-old chickens were intramuscularly immunized.A booster immunization was given by the same method as the first immunization 7 days later.The control groups were immunized with TE.At 28 days of age,chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered. Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that ACIs of experimental groups were beyond 160, which showed effective protection against avian coccidiosis.Preservation time and temperature had little effect on the immunizing efficacy of the vaccine in 6 months, indicating that the vaccine could be preserved for at least 6 months.The cross-protection of DNA vaccine pcDNA3.1-TA4-IL-2 was analysised.With 25μg of E.tenella DNA vaccine pcDNA3.1-TA4-IL-2,7-day-old chickens were intramuscularly immunized.A booster immunization was given by the same method as the first immunization 7 days later.The control groups were immunized with TE.At 28 days of age,chickens were challenged with sporulated oocysts of the corresponding Eimeria spp. JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered.Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that the vaccine could provide partial cross-protection against the challenge with E.necatrix and E.acervulina,but not with E. maxima.T cell epitopes of MZ5-7 gene and SO7 gene were predicted with DNAStar software. Results showed that the T cell epitopes of MZ5-7 gene located at the segments of 115-435 and 547-897,the first segment was named ml encoding 107 amino acids and the second segment was named m2 encoding 117 amino acids.The T cell epitopes of SO7 gene located at the segments of 7-291 and 370-609,the first segment was named s1 encoding 95 amino acids and the second segment was named s2 encoding 80 amino acids.Several pairs of primers were designed and m1,m2,s1and s2 segments were amplified by PCR with pMD18-T-SO7 and pMD18-T-MZ5-7 as templates.PCR products were cloned into pMD18-T vector.Identification showed that m1,m2,s1and s2 segments were cloned into pMD18-T vector successfully.m1,m2,s1 and s2 were inserted into pVAX1 vector with chIFN-γand chIL-2 as gene adjuvant,generating recombinant plasmids pVAX1-m1-m2-s1-s2-IFN口pVAX1-m1-m2-s1-s2-IL2,pVAX1-m1-s1-IFN口pVAX1-m1-s1-IL2,pVAX1-m1-s2-IFN口pVAX1-m1-s2-IL2,pVAX1-m2-s1-IFN口pVAX1-m2-s1-IL2,pVAX1-m2-s2-IFN口pVAX1-m2-s2-IL2,pVAX1-m1-m2-s1-s2,pVAX1-m1-s1,pVAX1-m1-s2,pVAX1-m2-s1, pVAX1-m2-s2,pVAX1-m1,pVAX1-m2,pVAX1-s1 and pVAX1-s2.After identified by restriction enzyme digestion,these recombinant DNA plasmids were extracted and injected into the leg muscle of 7 days old chickens.A week later the injected tissue was sampled respectively to check whether these DNA plasmid expressed or not by RT-PCR and Western-blot.The results indicated that aim genes could be successfully transcribed and expressed in injected tissues.Immune protection experiment was carried out with the 19 constructed plasmids. Nineteen experimental groups i.e.pVAX1-m1-m2-s1-s2-IFN口pVAX1-m1-m2-s1-s2-IL2, pVAX1-m1-s1-IFN口pVAX1-m1-s1-IL2,pVAX1-m1-s2-IFN口pVAX1-m1-s2-IL2, pVAX1-m2-s1-IFN口pVAX1-m2-s1-IL2,pVAX1-m2-s2-IFN口pVAX1-m2-s2-IL2, pVAX1-m1-m2-s1-s2,pVAX1-m1-s1,pVAX1-m1-s2,pVAX1-m2-s1,pVAX1-m2-s2, pVAX1-m1,pVAX1-m2,pVAX1-s1 and pVAX1-s2 groups were designed.The empty vector,challenged and unchallenged control groups were also designed.At the age of 14 days,experimental chickens were intramuscularly immunized with 100μg of the corresponding DNA vaccines.A booster immunization was given by the same method as the first immunization 7 days later.The empty control group was intramuscularly immunized with 100μg pVAX1.The other two control groups were intramuscularly immunized with TE.At 28 days of age,the chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge, all the chickens were slaughtered.Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that all the DNA vaccine constructed could induce effective protection against E.tenella.The DNA vaccine with multi-stage antigen genes of E.tenella could induce better protection than the ones with single stage.The DNA vaccines with cytokines could induce better protection than the without cytokines.Immunization with pVAX1-m1-m2-s1-s2-IFN口induced the most effective protection against E.tenella.更多还原