【作者】 王亚；

【导师】 余兴龙；

【作者基本信息】 湖南农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 猪霍乱沙门菌C500弱毒株是预防仔猪副伤寒的高效、安全的疫苗株。用其作载体,将含外源基因的真核表达质粒携带到宿主体内并表达外源蛋白,激活免疫系统,引起免疫反应。其独特的优点体现在既可作为针对沙门菌抗原的活疫苗,也可作为外源基因的活载体,并以其菌体成份的佐剂属性,增强机体的免疫应答反应。本实验利用DNA重组技术将鸡粒/巨噬细胞集落刺激因子基因（CGM-CSF）与鸡白细胞介素18基因（CIL-18）,克隆到真核表达载体pVAX1上,构建得到真核表达质粒pVAX1-CGMCSF:pVAX1-CIL18;pVAX1-CGMCSF/CIL-18。并将这3种质粒以及空质粒pVAX1分别转化C500菌。通过将C500（pVAX1-CGMCSF/CIL-18）和C500（pVAX1）体外培养和体内接种的方法,研究该真核表达质粒在体外和体内的稳定性,以及重组菌在体内的消长动态。同时将重组菌C500（pVAX1-CGMCSF/CIL-18）和C500（pVAX1）分别以10⁸、10⁹、10¹⁰CFU/只的剂量口服接种雏鸡,并在雏鸡体内传代接种的方法,研究该重组菌对雏鸡的安全性。结果显示重组菌在体外培养过程中,外界无抗性选择压力条件下,所携带质粒在重组菌中的稳定性也会随之降低。外界有抗性选择压力条件下,所携带质粒在重组菌传代过程中的稳定性于第8代后出现下降明显的趋势。在体内接种过程中,粪便分离菌通过酶切鉴定,血液,肝脏,脾脏,肠道粘膜所提DNA通过PCR鉴定均能获得清晰的目的条带,表明重组菌能将目的基因转运机体细胞内。将重组菌C500（pVAX1-CGMCSF/CIL-18）和C500（pVAX1）以10⁹CFU/只剂量口服接种雏鸡后,分别从脏器指数、鸡脾细胞增殖试验和碳粒廓清指数三个方面检测重组菌免疫后对雏鸡免疫功能的影响,结果显示了免疫功能指标均呈现重组菌C500（pVAX1-CGMCSF/CIL-18）组高于重组菌C500（pVAX1）组并显著高于对照组（p＜0.05）,表明重组菌接种后,进入体内释放出真核表达质粒,并能表达外源基因,促进雏鸡免疫器官的发育,提高机体的免疫功能。在此基础上,将C500（pVAX1）;C500（pVAX1-CGMCSF）;C500（pVAX1-CIL18）:C500（pVAX1-CGMCSF/CIL-18）分别免疫4组雏鸡,每组10只,于重组菌接种后1周各组以0.2ml/羽的剂量颈部皮下注射禽流感油乳剂灭活苗,设立PBS+灭活苗对照组,分别于免疫前、免疫后每隔1周翅静脉采血,通过间接血凝抑制试验检测血清抗体血凝抑制效价。结果显示了C500（pVAX1）;C500（pVAX1-CGMCSF）:C500（pVAX1-CIL18）;C500（pVAX1-CGMCSF/CIL-18）均能提高鸡群免疫灭活苗后的抗体效价,免疫后7天、14天、21天、28天C500（pVAX1-CGMCSF/CIL-18）组的血凝抑制效价均显著高于PBS对照组（P＜0.05）;C500（pVAX1-CGMCSF）组和C500（pVAX1-CIL18）组在免疫后7天、14天、21天血凝抑制效价显著高于PBS对照组（P＜0.05）,免疫后28天差异均不显著（P＞0.05）。表明用猪霍乱沙门菌C500弱毒株能将基因转运至机体内,携带的GM-CSF,IL-18可有效提高鸡的免疫功能和抗体水平,发挥了细胞因子GM-CSF和IL-18的免疫佐剂作用,为细胞因子之间的协同作用提供了实践依据。更多还原

【Abstract】 Attenuated choleraesuis salmonella C500 is efficient and secure vaccine strains for preventing piglet paratyphoid.As the vector,carrying eukaryotic expression plasmid which is containd foreign gene to the host and expressing exogenous protein will activate immune system and cause immune response.Its distinctive advantages are not only acting as live vaccine which is aiming at salmonella antigen,but also acting as live vector of foreign gene,and strengthen the organism’s immune response by its quality of adjuvant that it is the component of bacteria.This experiment clones the gene of chicken granulocyte / macrophage colonystimulating factor（CGM-CSF） and chicken interleukin-18（CIL-18） to the eukaryotic expression vector of pVAX1 by recombinant PCR technology,and with this construction, get the eukaryotic expression plasmids such as pVAX1-CGMCSF,pVAX1-CIL-18,pVAX1-CGMCSF/CIL-18.Getting eukaryotic expression plasmid into salmonella choleraesuis by the reorganization of conversion technology,we can construct these recombinant bacteria as C500（pVAX1）,C500（pVAX1-CGMCSF）,C500 （pVAX1-CIL18）,C500（pVAX1-CGMCSF/CIL-18）.This experiment researches the stability of the eukaryotic expression plasmid C500（pVAX1-CGMCSF/CIL-18）and C500（pVAX1）,the dynamics of recombinant bacteria in vivo by the method of getting C500（pVAX1-CGMCSF/CIL-18） and C500（pVAX1） foster in vitro and vaccinate in vivo.At the same time,this experiment also researches the security of the recombinant bacteria for young chicken by the method of inoculating from generation to generation in the young chicken that is vaccinating young chicken orally with the recombinant bacteria C500（pVAX1-CGMCSF/CIL-18） and C500（pVAX1） by the dosage as 10⁸,10⁹,10¹⁰ CFU per chicken separately.The results shows that the stability of the plasmid which is carried by the recombinant bacteria reduces when the recombinant bacteria is in the process of fostering in vitro and the condition of outside without resistance selection pressure.When the condition of outside is with resistance selection pressure,the stability of the plasmid carded by the recombinant bacteria reduces.Obviously after the 8th in the process of fostering from generation to generation. In the prosess of vaccination in vivo,both the bacteria isolated from.Faeces that identified through the restrinction endonuclease analysis and the DNA extracted from the blood,liver,spleen and intestinal mucosa that identified through the PCR can get clear purpose strip.This shows that the recombinant bacteria can transferre the goal gene to the cells in organisms.In view of the cytokines both GM-CSF and IL-18 are immunoregulatory factors and they can strengthen the immune function,we vaccinate the young chicken orally with the recombinant bacteria C500（pVAX1-CGMCSF/CIL-18） and C500（pVAX1） 10⁹CFU per chicken.Then we detect the influence of immune function from tree aspects that organ index,chicken spleen cells proliferation test and carbon clearance index.The results show that the immune function indicators of the group which is vaccinated C500（pVAX1-CGMCSF/CIL-18） are higer than the group which is vaccinated C500（pVAX1）,and significantly higher than the control group（p＜0.05）. This indicates that the recombinant bacteria vaccinated and after they get into the immune organs,they can release eukaryotic expression plasmid,express foreign gene, stimulate the immune function that against salmonella carrier and foreign gene,and promote the young chickens’ immune organs growing.On this basis,we vaccinate 4 groups with C500（pVAX1）,C500（pVAX1-CGMCSF）, C500（pVAX1-CIL18）,C500（pVAX1-CGMCSF/CIL-18） separately and every group is 10 chickens.Then we take blood from wing vein every week respectively before immunity and after immunity,and detect the hemagglutination inhibition titer of serum antibody through indirect hemagglutination inhibition test.The result shows that C500（pVAX1）,C500（pVAX1-CGMCSF）,C500（pVAX1-CIL18）,and C500 （pVAX1-CGMCSF/CIL-18） can all raise the Antibody titer after the chickens vaccinated inactivated vaccine.This shows that the recombinant bacteria using attenuated salmonella choleraesuis C500 as vector can raise the young chickens’ antibody level which is against the avian influenza oil-emulsion inactivated vaccine effectively,it plays the function of cytokines GM-CSF and IL-18 as immune adjuvant,and it provides practice foundation for the synergy between cytokines.更多还原