猪多杀性巴氏杆菌皮肤坏死毒素亚单位蛋白的制备及免疫原性研究  

Study on Preparation and Immunogenicity of Subunit Proteins of Pasteurella multocida Toxin from Pigs

作　　者：何海 郝成武 凌晨 张飞 候凤 贺笋 HE Hai;HAO Chengwu;LING Chen;ZHANG Fei;HOU Feng;HE Sun(Tecon Bio-technology Co.,Ltd.,Urumqi 830011,China)

机构地区：[1]天康生物股份有限公司

出　　处：《中国畜牧兽医》2019年第11期3396-3403,共8页China Animal Husbandry & Veterinary Medicine

基　　金：天康生物技术中心项目(2011005)

摘　　要：为获得针对猪萎缩性鼻炎主要病原体——多杀性巴氏杆菌皮肤坏死毒素的高免疫原性抗原,本试验构建、表达并验证该毒素的亚单位蛋白抗原,将猪多杀性巴氏杆菌毒素PMT基因与pMD19-T载体进行连接、转化,经序列分析鉴定后,分别使用Bam HⅠ、Hin dⅢ与BlpⅠ限制性内切酶将其酶切为3个基因片段。将片段1(Tox1)、片段2(Tox2)和片段3(Tox3)分别亚克隆至原核表达载体pET32-b、pET32-a和pET32-b中,构建3个重组表达载体。将重组表达载体转化E.coli BL21(DE3)感受态细胞,经IPTG诱导后,分别进行SDS-PAGE与Western blotting检测,并使用小鼠与豚鼠初步研究其免疫原性。结果显示,构建的3个基因片段长度分别为776、409与410 bp,与GenBank中相关序列具有高度同源性;3个蛋白片段表达正常,表达量分别达到379.95、447.62与459.82μg/mL,SDS-PAGE验证条带分别为75、77与53 ku;因3个片段位置不同,仅Tox3有Western blotting检测条带,与理论预测相符;使用3种表达蛋白免疫小鼠与豚鼠后,二免后14 d,血清经试剂盒检测阳性率达到100%,对二免后14 d小鼠攻毒保护率达到93%。本研究成功构建了PMT的3个亚单位活性片段,且具有较好的免疫原性。In order to obtain the high immunogenicity antigen of the main pathogen of porcine atrophic rhinitis,Pasteurella multocida toxin,the subunit protein antigen of the Pasteurella multocida toxin was constructed,expressed and verified.The PMT gene of porcine Pasteurella multocida toxin was linked and transformed to pMD19-T vector,and identified by sequence analysis.Bam HⅠ,Hin dⅢand BlpⅠrestriction endonucleases were used to digest three gene fragments.Fragment 1(Tox1),fragment 2(Tox2)and fragment 3(Tox3)were subcloned into prokaryotic expression vectors pET32-b,pET32-a and pET32-b,respectively,and three recombinant expression vectors were constructed.The recombinant expression vector was transformed into E.coli BL21(DE3)competent cells.After induction by IPTG,SDS-PAGE and Western blotting were performed respectively.The immunogenicity of the recombinant expression vector was preliminarily studied in mice and guinea pigs.The results showed that the length of three gene fragments were 776,409 and 410 bp,which were highly homologous with the related sequences in GenBank.Three protein fragments were expressed normally,with the expression levels reaching 379.95,447.62 and 459.82μg/mL,respectively.The SDS-PAGE validation bands were 75,77 and 53 ku,respectively.Because of the different positions of the three fragments,only Tox3 had Western blotting detection bands,which was consistent with the theoretical prediction.After immunizing mice and guinea pigs with three expressed proteins,the positive rate of serum was 100%on the 14th day after immunization,and the protected rate was 93%to mice on the 14th day after immunization.In this study,three subunit active fragments of PMT were successfully constructed,and they had good immunogenicity.

关 键 词：多杀性巴氏杆菌皮肤坏死毒素 亚单位活性片段 免疫原性 

分 类 号：S858.28[农业科学—临床兽医学]