【作者】 李鹏；

【导师】 陈金顶；

【作者基本信息】 华南农业大学 ， 兽医硕士（专业学位）， 2017， 硕士

【摘要】 猪传染性胸膜肺炎(Porcine contagious pleuropneumonia,PCP)是影响全球养猪业的重要细菌性疾病之一,它是一种由胸膜肺炎放线杆菌引起的以肺脏纤维素性胸膜肺炎为主要病变的呼吸道传染病。该病目前在世界范围内广泛流行,给集约化养猪业造成严重的经济损失。我国当前预防该病的主要手段是接种灭活疫苗,但是由于该致病原血清型较多、毒力因子复杂等原因,该疫苗的保护效果不好。因此,开发新型、高效的疫苗已成为防控该病的热点。溶血外毒素和外膜蛋白作为APP的重要毒力因子和免疫原,在该病的感染致病过程中发挥着重要的作用。为此,本研究制备4种外毒素蛋白和外膜蛋白为组分的亚单位疫苗,开展其免疫效果研究,具体如下:应用PCR的方法从商品化灭活疫苗中扩增ApxⅢA和ApxⅣA基因,并将其克隆到表达载体pET-32a中,再以PCR、双酶切鉴定和DNA测序验证重组质粒pET-ApxⅢA、pET-ApxⅣA。将重组质粒转化进入大肠杆菌BL21(DE3)感受态细胞中,在IPTG作用下诱导表达,经SDS-PAGE和Western blot检测重组蛋白,结果显示rApxⅢA和r ApxⅣA大小分别约为50和45 k D,并证明重组蛋白具有较好的反应原性。在实验室前期研究的基础上,用获得的rApxⅢA和r ApxⅣA蛋白,制备成油佐剂免疫小鼠进行免疫实验,免疫分组如下:Ⅰ组为PBS阴性对照,Ⅱ组为商品化三价灭活疫苗阳性对照,Ⅲ组为三价灭活疫苗和白油佐剂乳化的rApxⅠA、r ApxⅡA、rApxⅢA、r ApxⅣA和rOMP蛋白混合疫苗,Ⅳ组为弗氏佐剂乳化的rApxⅠA、rApxⅡA、r ApxⅢA、r ApxⅣA和rOMP蛋白,Ⅴ组为白油佐剂乳化的rApxⅠA、rApxⅡA、rApxⅢA、r ApxⅣA和rOMP蛋白。每组均分三次免疫,每次间隔2周,每次免疫1周后对小鼠采血,以间接ELISA测定小鼠血清特异性抗体变化,第三次免疫1周后对每组小鼠分别以APP1型(4.56×10~7cfu)进行攻毒。结果显示疫苗Ⅳ、Ⅴ组的抗体水平基本一致,高于Ⅱ组,高于Ⅰ组,疫苗Ⅳ组和V组对APP1型攻毒保护作用(4/10)相同,高于Ⅲ组(2/10)和Ⅰ组(0/10),低于Ⅱ组(5/10)。表明本研究中的亚单位疫苗对小鼠是安全的,具有较好的保护效果,且白油佐剂和弗氏佐剂配制的亚单位疫苗都能刺激小鼠产生较高的抗体水平。本研究为PCP的新型疫苗的的研制提供了参考。更多还原

【Abstract】 Porcine contagious pleuropneumonia(PCP)is one of the most important bacterial diseases affecting the global pig industry.It is a respiratory disease caused by Actinobacillus pleuropneumoniae(APP)with lung fibrous pleural pneumonia as the main lesion.The disease is currently widespread in the world,caused serious economic losses to the intensive pig industry.C urrently,the prevention of the disease in C hina is inactivated vaccine,but because of the different serotype,complexing virulence factors and other reasons the inactivated vaccine can not provide completely protective effect.Therefo re,development of new and efficient vaccine has become hot spots in disease research.As important virulence factors and immunogen of APP,hemolytic exotoxin and outer membrane proteins play an important role in the pathogenesis and the process of the disease.Based on this,the study takes four kinds of exotoxin and outer membrane protein as a component subunit vaccine to explore its immune effect,as follows:The ApxⅢA and ApxⅣA genes were amplified from the commercial inactivated vaccine by PCR and cloned into the expression vector p ET-32 a.The recombinant plasmid p ET-ApxⅢA and p ET-ApxⅣA was confirmed by PCR,double digestion,.DN A sequencing.The results showed that the recombinant expression plasmid was successfully constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)competent cells and induced by IPTG.The recombinant protein was detected by SDS-PAGE and Western blot.The results showed that r ApxⅢA and r ApxⅣA were about 50 and 45 k D Western blot analysis showed that the recombinant protein had good reactogenicity.Based on the preliminary study of the laboratory,the mice immunized with the oil adjuvant obtained r ApxⅢA and r ApxⅣA protein.The immunization group was as follows: Group Ⅰ was treated with PBS as blank control group.Group II commercialized trivalent inactivated vaccine was positive control group.Group III was a trivalent inactivated vaccine and white oil adjuvant emulsified r ApxⅠA,r ApxⅡA,r ApxⅢA,r ApxⅣA and r OMP protein mixed vaccine.The r ApxⅠA,r ApxⅡA,r ApxⅢA,r ApxⅣA and r OMP proteins emulsified with Freund’s adjuvant were used as test group,r ApxⅠA,r ApxⅡA,r ApxⅢA,r ApxⅣA,and r OMP were tested in group V.Each group was given three immunization,each interval for two weeks,and the mice were given blood after one week of each immunization,i ELISA was used to determine the change of serum specific antibodies in mice.Each group of mice were treated with APP1(4.56 x 107cfu)one week after the third immunization.Results showed that the vaccine Ⅳ,Ⅴ group antibody levels are basically identical which was higher than group Ⅱ,and higher than group Ⅰ.Group Ⅳ and group V had the same protective effect on APP1 challenge(4/10),which was higher than group Ⅲ(2/10)and group I(0/10),but lower than group Ⅱ(5/10).It showed that the subunit vaccine in this study was safe for mice and had good protective effect.Both the white oil adjuvant and the Freund’s adjuvant could stimulate the antibody production.This study provides a reference for PCP’s new vaccine and diagnostic methods.更多还原