猪APP rOmpP2诱导小鼠抗攻击感染的免疫保护力研究

发布时间：2018-04-28 21:37

本文选题：胸膜肺炎放线杆菌(APP) + 重组外膜蛋白(rOmpP)　；参考：《农业生物技术学报》2017年02期

【摘要】：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Gen Bank中已报道的APP Omp P2基因序列设计引物对,以APP的基因组为模板PCR扩增Omp P2,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p ET32a(+)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 k D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,Ig G)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+30μg r Om P2及铝佐剂+10μg 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。
[Abstract]:Porcine contagious pleuropneumoniae is a contact respiratory disease caused by Actinobacillus pleuropneumoniae APP (Actinobacillus pleuropneumoniae). In order to detect the immune protection of porcine APP recombinant outer membrane protein P2(recombinant outer membrane protein P2rOmpP2, primer pairs were designed according to the sequence of APP Omp P2 gene reported in Gen Bank. Using APP genome as template, Omp P2was amplified by PCR, sequenced, and then registered in NCBI (accession number: KM357903G). The gene was composed of 1 131bp and encoded a 376-amino acid protein, which was an outer membrane pore protein. The encoding protein has high homology among different serotypes of APP. ROmpP2 was subcloned into pET32a () vector and transformed into Escherichia coli BL21DE3G. The expression of APP rOmpP2 protein was induced by isopropyl 尾 -D-thiogalactopyranoside (Isopropyl 尾 -D-thiogalactopyranoside). The expression of APP rOmpP2 protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGEG) and identified by Western blot. The results showed that there was a specific band at 42 KD, and the rOmpP2 expressed by the recombinant strain could be recognized by the corresponding positive serum. The immunoglobulin (G(immunoglobulin) IgG antibody titer was detected by enzyme linked immunosorbent assay (Elisa). The results showed that the survival rates of 50 渭 g rOmpP2, 30 渭 g r Om P2 and 10 渭 g rOmpP2 groups were 50% and 10%, respectively. The subcutaneous injection of .rOmpP2 could induce immune response and higher immune protection against aggressive infection in mice. The results provide basic information for screening molecular vaccine against swine infectious pleuropneumonia.
【作者单位】：湖南农业大学动物医学院;
【基金】：教育部留学回国科研启动基金(No.GT0096)
【分类号】：S858.28

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