貂源绿脓杆菌山东分离株exoA-fliC基因的原核表达及其免疫原性研究  

Prokaryotic Expression and Immunogenicity of exoA-fliC fusion Gene of Pseudomonas Aeruginosa from Mink in Shandong Province

作　　者：秦晓冰[1] 马凤芹[1] 单虎[1] QIN Xiao-bing;MA Feng-qin;SHAN Hu(Shandong Key Laborary of Preventive Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学山东省预防兽医学重点实验室,山东青岛266109

出　　处：《中国兽医杂志》2018年第8期50-53,共4页Chinese Journal of Veterinary Medicine

基　　金：山东省兽药诊断试剂工程技术研究中心;山东省高等学校优势学科人才团队培育计划

摘　　要：采用重叠PCR方法扩增水貂绿脓杆菌山东分离株(PASD01株)的exoA与fli C基因,并将两段基因嵌合后,克隆到表达载体p ET-30a上,转化到宿主菌BL21(DE3)中诱导表达,并对表达产物进行免疫原性分析。结果表明,重组嵌合蛋白在37℃,0. 8 mmol/L IPTG诱导7 h时表达量最高;经SDS-PAGE和Western Blot分析,该蛋白在约68 k D处出现特异性条带,与预期结果一致;该重组嵌合蛋白免疫小鼠后,最高血清效价可达1∶640 000。结论:水貂绿脓杆菌exoA-fli C嵌合蛋白具有良好的免疫原性,为其进一步发展成为新型亚单位疫苗来抵抗水貂出血性肺炎奠定基础。ExoA and fliC gene fragments from the genome of P. aeruginosa isolated from mink in Shandong （ PASD01 strain）were amplified respectively. Then, overlapping PCR was used to get exoA-fliC fusion gene which was cloned into pET-30a prokaryoticexpression vector. The recombinant vector was transformed into E coli. BL21 （DE3） competent cells were used for expression. LaterKuming mice were immunized with the refolding protein to analyze its immunogenicity. We found that the constructed recombinantvector by restriction endonuclease digestion was consistent with the expected size about 1722 bp, and the optimized condition for ex-pressing target protein was induced 7h at 37℃ with 0. 8 mmol / L IPTG. Approximately 68 KD recombinant protein band was observedby both SDS-PAGE and Westernblot, and 1：640000 sera titer stimulated by the aimed protein was detected by indirect ELISA in theimmunized mice. So exoA-fliC recombinant proteins as a new subunit vaccine agnist mink hemorrhagic pneumonia is promised to bedeveloped against mink hemorrhagic pneumonia.

关 键 词：水貂绿脓杆菌 exoA-fliC嵌合基因 原核表达 免疫原性 

分 类 号：S858.292[农业科学—临床兽医学]