副鸡禽杆菌体内诱导交叉保护性抗原的筛选及鉴定

Avibacterium paragallinarum is the causative agent of infectious coryza, one of the important pathogens that cause huge economic losses at poultry industry. Vaccination is the main way to control it. It has been confirmed that the vaccine made from killed Avibacterium paragallinarum lacks of cross-protection ability among serotypes. However, the experimentally infected chickens can produce cross-immunity against the strains of different serum. And, the recovery chickens after infected with field strains have strong resistance against re-infection of any strains. This suggests that some important antigens of the Avibacterium paragallinarum are specially expressed in vivo, and some of in vivo induced antigens (IVIA) are important cross protective antigens against Avibacterium paragallinarum. This project intends to build a phage expression library of genomic DNA of Avibacterium paragallinarum. According to the principle of in vivo induced antigen technology, a database of coded genes of IVIA would be gained by library screening. The function of IVIA will be predicted and analyzed by professional bioinformatics softwares. Their immunogenicities and protective efficacy will be tested in animal assays. In order to analyze the roles of their coding protein further more, mutant strains of in vivo induced gene deletion will be constructed. Changes of their biological activities will be checked by testing them with their parental strains together. This study has an important significance to draw an in vivo-induced gene expression profile of Avibacterium paragallinarum, to clarify the function of the in vivo induced antigens. At the same time, the obtained cross protective antigens might lay the foundation for the development of new Infectious Coryza vaccines.

副鸡禽杆菌是鸡传染性鼻炎的病原菌，是危害养鸡生产的重要病原之一。免疫接种是其主要防治措施，但副鸡禽杆菌全菌灭活疫苗缺乏血清型间的交叉保护，而实验感染鸡可以产生对其他血清型菌株的交叉免疫力。并且，田间感染康复鸡对再次感染也有很强的抵抗力。这表明该病原体某些重要抗原的编码基因只在体内表达,某些体内诱导抗原是副鸡禽杆菌重要的交叉保护性抗原成分。本项目拟构建副鸡禽杆菌基因组DNA的噬菌体文库，根据体内诱导抗原技术的原理，筛选获得副鸡禽杆菌体内诱导基因数据。应用生物信息学软件工具分析预测体内诱导抗原的功能；克隆表达并动物攻毒保护试验检测疑似交叉保护性抗原的免疫原性；通过构建体内诱导基因缺失突变菌株，比较与亲本株生物活性的差异,进一步分析该基因的功能，并探讨其编码蛋白的作用。本研究对绘制副鸡禽杆菌体内诱导基因谱、阐明体内诱导抗原的功能具有重要意义。交叉保护性抗原可为副鸡禽杆菌新型疫苗的研究奠定基础。

副鸡禽杆菌（Avibacterium paragallinarum，Apg）是鸡传染性鼻炎的致病菌，主要引起鸡的生长发育受阻，产蛋量下降，给养鸡业带了相当大的经济损失。病原菌体内诱导基因的研究是探索该病防治措施的重要基础。.副鸡禽杆菌侵入宿主后，为了适应体内的环境，会启动一系列基因的表达，这些体内诱导基因有可能是引起疾病的关键基因,有可能作为候选的免疫及药物分子新靶位。.本研究首先提取Apg的基因组DNA，用Sau3A I 酶切后回收片段插入到pET系统表达载体中，构建副鸡禽杆菌的全基因组表达文库。应用体内诱导抗原技术(In vivo induced antigen technology ,IVIAT) 的原理，先用IPTG 诱导文库表达蛋白，然后用经体外培养的副鸡禽杆菌菌株和大肠杆菌BL21(DE3)吸附过的鸡副鸡禽杆菌血清对该文库进行初筛和复筛，对筛选得到的阳性克隆提取质粒用T7启动子引物测序，测得的序列在NCBI上比对后确定开放阅读框。.结果显示，本实验构建的副鸡禽杆菌基因组表达文库大小为6×103，文库重组质粒含有率大于80%,达到了理论要求。经过筛选、测序和比对本实验最终确定了5个开放阅读框。有一个表达为转运谷氨酰还原酶，一个转录终止因子和荚膜合成域2基因簇，还有两个表达为保守假想蛋白。.在此基础上, 以筛选获得的荚膜合成域基因和aroA基因为研究对象构建基因缺失载体，并将该载体分别电转入到A型副鸡禽杆菌野生菌株，通过同源重组获得突变菌株，对突变菌株进行了鉴定和生物学特性分析。结果表明，突变菌株细菌形态未见明显变化，生长曲线提示其生长未受显著影响，但致病能力较低，初步验证了体内诱导抗原的作用。为研究副鸡禽杆菌在宿主体内生存和致病关键基因奠定了基础，也为鸡传染性鼻炎防治研究提供了思路。