猪APP rOmpP2诱导小鼠抗攻击感染的免疫保护力研究

摘要

Porcine contagious pleuropneumonia (PCP) is a respiratory disease caused by Actinobacillus pleuropneumoniae (APP) infection,which is one of the 5 major epidemics in the world's pig industry.Based on APP OmpP2 sequences in GenBank,the primers were designed.APP OmpP2 was amplified by PCR from APP genome,sequenced and submitted to NCBI (Access No.KM357903).Bioinformatics analysis showed that the APP OmpP2 gene was composed of 1 131 bp,encoding a 376 amino acid protein.This protein was a porin protein with high homology among different APP serotypes.APP OmpP2 gene was subcloned into pET32a (+) to construct recombinant plasmid pET-OmpP2,rOmpP2 expression in Escherichia coli BL21 was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG),and determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.The results showed that rOmpP2 with 42 kD was detected.Animal experiment was conducted by BALB/c mice (Mus musculus).The serum immunoglobulin G (IgG) level was detected by enzyme-linked immuno sorbent assay (ELISA).The results showed that survival rate of infected mice with aluminium adjuvant+50 μg rOmpP2,aluminium adjuvant+30 μg rOmpP2 and aluminium adjuvant+ 10 μg rOmpP2 were 50％,30％ and 10％,respectively,and all mice died in adjuvant group and control group.The surviving mice slowly returned to normal health status.Mice that were subcutaneously immunized with rOmpP2 showed an partial protection against the challenge with APP.This study provides basic data for screening new molecular vaccines of porcine contagious pleuropneumonia.%猪传染性胸膜肺炎(porcine contagious pleuropneumoni,PCP)是由胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)感染猪(Sus scrofa)引起的一种接触性呼吸道疾病,是世界养猪业的五大疫病之一.为检测猪APP重组外膜蛋白P2 (recombinant outer membrane protein P2,rOmpP2)的免疫保护作用,本研究根据GenBank中已报道的APP OmpP2基因序列设计引物对,以APP的基因组为模板PCR扩增OmpP2,测序后在NCBI登录(登录号:KM357903),并进行生物信息学分析,该基因由1 131 bp组成,编码一个含376个氨基酸的蛋白质,为一外膜孔道蛋白,该编码蛋白在APP不同的血清型之间具有很高同源性.将rOmpP2亚克隆至pET32a(+)载体,转化大肠杆菌(Escherichia coli) BL21 (DE3),用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达APP rOmpP2蛋白,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)检测以及Western blot鉴定,结果表明,在42 kD处出现特异性条带,重组菌表达的rOmpP2能够被相应的阳性血清识别.动物保护实验采用BALB/c小鼠(Mus musculus)进行,免疫球蛋白G(immunoglobulin G,IgG)抗体滴度采用酶联免疫吸附测定(enzyme-linked immuno sorbent assay,ELISA)检测.结果表明,铝佐剂+50μg rOmpP2、铝佐剂+30 μg rOmP2及铝佐剂+10 μg rOmpP2组中,小鼠的存活率分别为50％,30％和10％,佐剂组和生理盐水对照组全部死亡,存活小鼠精神状况慢慢恢复正常,并采食.rOmpP2皮下免疫注射可诱导小鼠产生免疫应答和较高的抗攻击感染的免疫保护力.研究结果为进一步筛选猪传染性胸膜肺炎分子疫苗提供了基础资料.