摘要
为提高滴鼻免疫条件下猪支原体肺炎活疫苗的免疫效果,本研究设计引物扩增猪肺炎支原体P97R1基因,融合至霍乱毒素B亚单位(CTB)基因下游,构建了重组质粒p ET-CTB-P97R1。重组菌经IPTG诱导表达、分离纯化得到r CTB-P97R1重组蛋白。利用体外神经节苷脂(GM1)结合试验检测r CTB-P97R1的佐剂活性。随后将r CTB-P97R1与活疫苗混合后滴鼻免疫小鼠,免疫后采血和收集小鼠支气管肺泡灌洗液(BALF),分别检测其中的特异性Ig G抗体和s Ig A抗体的水平评价免疫效果。结果显示,r CTB-P97R1可以特异性结合GM1,具有生物学活性。小鼠免疫后的血清Ig G抗体显著高于活疫苗对照组(p<0.01),同时产生了高水平的P97R1特异性Ig G抗体(p<0.01);小鼠BALF中的s Ig A抗体也显著高于活疫苗对照组(p<0.05)。结果表明,r CTB-P97R1能够显著增强经滴鼻免疫的猪支原体肺炎活疫苗的免疫效力,同时可提升P97R1的免疫应答,有望作为经黏膜免疫的猪支原体肺炎活疫苗的新型佐剂。
To enhance the immunostimulating effect of the Mycoplasma hyopneumoniae live vaccine via intranasal injection,the P97 R1 gene of Mycoplasma hyopneumoniae was amplified and fused to the downstream of the CTB gene to construct a recombinant expressing plasmid pETCTB-P97 R1 and transformed to E.coli. The recombinant E.coli was induced by IPTG to express the fusion recombinant protein CTB-P97 R1(rCTB-P97 R1), which was purified by affinity chromatography. The adjuvant bioactivity of rC TB-P97 R1 was detected by the monosialoganglioside(GM1)-ELISA assay. BALB/c mice were immunized with the rC TB-P97 R1 coupled with live vaccine through intranasal route. Serum and bronchoalveolar lavage fluid(BALF) samples were collected and detected for specific IgG and sI gA antibodies against the whole Mycoplasma protein or P97 R1 protein. The results showed that the rC TB-P97 R1 was able to specifically bind to GM1, indicating a well biological activity in vitro. In immunization experiment, high levels of antibodies against the whole Mycoplasma protein and P97 R1 were detected after the immunization inthe serum of the mice immunized with CTB-P97 R1-coupled live vaccine(p<0.01), compared with the mice immunized with live vaccine alone. The sI gA antibody against the whole Mycoplasma protein in BALF of the mice immunized with rC TB-P97 R1-coupled live vaccine was significantly higher than that of the mice in immunized group with live vaccine alone(p<0.05). In conclusion, rC TB-P97 R1 enhanced the immunostimulating effect of the live vaccine through intranasal route, as well as the immune response to the key antigen P97 R1. The data indicated its potential to be used as a new adjuvant for the mucosal immunity live vaccine against M.hyopneumoniae.
作者
韦艳娜
张珍珍
王丽
王佳
陈蓉
冯志新
邵国青
熊祺琰
WEI Yan-na;ZHANG Zhen-zhen;WANG Li;WANG Jia;CHEN Rong;FENG Zhi-xin;SHAO Guo-qing;XIONG Qi-yan(Institute of Veterinary Medicine,Jiangsu Academy of Agriculture Sciences,Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture,Nanjing 210014,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2019年第1期74-79,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家重点研发计划(2017YFD0501604)
