胸膜肺炎放线杆菌血清型特异的疫苗候选基因的筛选及痤疮丙酸杆菌异源免疫

Screening of Serotype-specific Vaccine Candidate Gene of Actinobacillus pleuropneumoniae and Heterologous Immunization with Propionibacterium acnes

目的筛选猪胸膜肺炎放线杆菌(APP)1型与5型特异的疫苗候选基因,并研究痤疮丙酸杆菌对猪传染性胸膜肺炎的异源免疫。方法采用cDNA代表性差异分析、大肠杆菌核糖体展示、免疫筛选和RT-PCR技术筛选APP1型和5型血清型特异的疫苗候选基因,进行克隆、测序及同源序列分析。并用与APP疫苗候选基因具有同源序列的痤疮丙酸杆菌(PA)免疫小鼠,研究其对APP感染的异源免疫。结果获得血清1型APP6条疫苗候选基因,2条为未知基因,2条与PA同源性为98%,2条与人cDNA有同源性。获得血清5型APP7条疫苗候选基因,其中2条为未知基因,2条与PA同源性分别为93%和100%。用PA全菌免疫小鼠,产生的抗血清可与1型和5型APP发生强的免疫反应,ELISA检测效价达1∶3200;脾淋巴细胞检测结果显示,免疫组CD3+、CD4+T细胞的百分率及CD4+/CD8+比值与对照组相比均升高,两组CD3+T细胞百分率差异有显著意义。以10倍LD501型和5型APP分别感染PA免疫小鼠,小鼠存活率分别为95%和90%,并且在攻毒后第15天体内APP可全部被清除。结论1型与5型APP存在不同的疫苗候选基因,痤疮丙酸杆菌与之存在共同抗原,能够产生交叉免疫应答,对血清1型和5型APP的感染具有预防作用。

Objective To screen the serotypes 1- and 5-specific vaccine candidate gene of Actinobacillus pleuropneumoniae （APP） and study the heterologous immunization against swine pleuropneumoniae with Propionibacterium acnes. Methods The serotypes 1- and 5-specific vaccine candidate genes of APP were screened by cDNA representational difference analysis （cDNA-RDA）, E. coli ribosome display, immune screening and RT-PCR, then cloned, sequenced and analyzed for homology. Immunize mice with Propionibacterium acnes（PA） of which the sequence was homologous to that of candidate gene of APP vaccine to explore the heterologous immunization against APP infection. Results Six APP serotype 1-specific vaccine candidate gene fragments were obtained, of which 2 were unknown, 2 were homologous to human cDNA, and either the other 2 showed a homology of 98% to PA. However, of the obtained 7 APP serotype 5-specific vaccine candidate gene fragments, 2 were unknown, and 2 showed homologies of 93% and 100% to PA respectively. The antiserum of mice immunized with PA showed strong immune reactions with both APP serotypes 1 and 5, and reached an ELISA titer of 1 ∶ 3 200. Compared with those of control, the percentage of CD3^＋ T lymphocytes of mice immunized with PA increased significantly, and the percentage of CD4^＋ T lymphocytes as well as CD4^＋ / CD8^＋ ratio also increased. The survival rates of mice immunized with PA were 95% and 90% respectively after challenge with APP serotypes 1 and 5, each at a dosage of 10 ×LD50. No APP was isolated from the organs of immunized mice on day 15 after challenge. Conclusion The vaccine candidate genes of APP serotypes 1 and 5 are different. However, the common antigen of APP and PA was proved in this study. The cross protection highlights the possibility of using PA vaccine for preventing infection with APP serotypes 1 and 5.