新型重组减毒鸡白痢沙门菌平衡致死系统的构建及其特性
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- 英文论文题名：Construction and characterization of a novel balanced lethal system based on attenuated Salmonella pullorum △crp△asdC79-13(pcD-asd)
- 论文作者：翟崇凯 ; 郁川 ; 程相朝 ; 余祖华 ; 何雷 ; 廖成水 ; 张春杰 ; 李银聚 ; 田文静
- 英文论文作者：ZHAI Chongkai ; YU Chuan ; CHENG Xiangchao ; YU Zuhua ; HE Lei ; LIAO Chengshui ; ZHANG Chunjie ; LI Yinjun ; TIAN Wenjing ; The Key Lab of Animal Disease and Public security ; Henan University of Science and Technology ; Luoyang Vocational & Technical College
- 年：2016
- 作者机构：河南科技大学动物疫病与公共安全重点实验室;洛阳职业技术学院;
- 论文关键词：减毒沙门菌 ; 无抗性基因 ; 平衡致死系统 ; 活疫苗载体
- 英文论文关键词：attenuated Salmonella ; non-resistance ; balance-lethal system ; live vaccine vector
- 会议召开时间：2016-11-04
- 会议录名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会论文集
- 语种：中文
- 分类号：S852.61
- 学会代码：ZGXJ
- 会议名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会
- 会议地点：中国山东青岛
- 主办单位：中国畜牧兽医学会动物微生态学分会
- 学会名称：中国畜牧兽医学会
- 页数：1
- 文件大小：87k
- 原文格式：O
- 会议级别：全国
摘要
为开发新型重组减毒鸡白痢沙门菌口服活疫苗载体。本研究以pcDNA3.1-Zeo(+)质粒为基础,用沙门菌asd基因替代其氨苄抗性基因4mp,构建不含抗性基因的真核互补质粒pcD-asd;再将互补质粒pcD-asd电转入沙门菌△crp△asdC79-13,构建△crp△asdC79-13(pcD-asd)平衡致死系统,研究其生物学特性,进一步将报告基因EGFP克隆入pcD-asd,构建重组菌株△crp△asdC79-13(pcD-asd-EGFP),感染鸡胚成纤维(CEF)细胞,Western Blot分析该系统递呈外源抗原的能力。PCR、酶切及测序结果表明△crp△asdC79-13(pcD-asd)平衡致死系统构建成功;生物学特性鉴定结果表明,其血清型与亲本株△crp△asdC79-13及野生株C79-13保持一致;其生化特性与亲本株基本相近,但与野毒株相比发生明显变化;生长速度也更为缓慢,对氨苄抗生素敏感;重组菌株△crp△asd C79-13(pcD-asd)的LD_(50)较野生株C79-13降低了3.4×10~4倍;Western Blot结果发现,重组菌株感染CEF细胞后,可表达大小约为27KDaEGFP蛋白。以上结果证实,本研究成功构建了新型重组减毒鸡白痢沙门菌△crp△asdC79-13(pcD-asd)株,其能够有效递呈外源抗原,该重组菌株有潜力作为安全、稳定、高效表达外源基因的口服重组活疫苗载体。
In order to develop a new recombinant attenuated Salmonella pullorum as oral live vaccine vector.A new non-antibiotic eukaryotic recombinant plasmid renamed as pcD-asd was constructed by replacing the ampicillin resistance gene Amp with the asd gene of Salmonella on the basis of pcDNA3.1-Zeo(+) plasmid.The complementary plasmid pcD-asd was electrotransformed into △crp△asdC79-13.△crp△asdC79-13(pcD-asd) balanced lethal system was constructed,and then the system of biological characteristics was further researched.The reporter gene-enhanced green fluorescent protein(EGFP) gene was cloned into pcD-asd and the recombinant strain △crp△asdC79-13(pcD-asd-EGFP)was constructed,infection of chick embryo fibroblast(CEF) cells.The ability of delivery foreign antigens was tested via Western blot analysis.The results of PCR,enzyme digestion and sequencing showed that the △crp△asd C79-13(pcD-asd) balanced lethal system was constructed successfully.Biological characteristics showed that the serotype of△crp △asdC79-13(pcD-asd) was identical to △crp△asdC79-13 and C79-13;the biochemical characteristics of AcrpAasdC79-13(pcD-asd) were basically the same with △crp△asdC79-13 strain,however,it has a significant different with the wild strain C79-13;growth rate was also more slowly;it was sensitive to ampicillin;the median lethal dose reduces the 3.4×10~4 times of C79-13.Western blot analysis showed that the size of the foreign protein was about 27 KDa,when the recombinant strain infeced CEF cells.These results demonstrated that the new recombinant Salmonella pullorum △crp△asdC79-13(pcD-asd),which can present exogenous antigenwas effectively and have great potential be used as an oral live vaccine vector for expressing foreign genes efficiently and safety and stably,was constructed successfully.
In order to develop a new recombinant attenuated Salmonella pullorum as oral live vaccine vector.A new non-antibiotic eukaryotic recombinant plasmid renamed as pcD-asd was constructed by replacing the ampicillin resistance gene Amp with the asd gene of Salmonella on the basis of pcDNA3.1-Zeo(+) plasmid.The complementary plasmid pcD-asd was electrotransformed into △crp△asdC79-13.△crp△asdC79-13(pcD-asd) balanced lethal system was constructed,and then the system of biological characteristics was further researched.The reporter gene-enhanced green fluorescent protein(EGFP) gene was cloned into pcD-asd and the recombinant strain △crp△asdC79-13(pcD-asd-EGFP)was constructed,infection of chick embryo fibroblast(CEF) cells.The ability of delivery foreign antigens was tested via Western blot analysis.The results of PCR,enzyme digestion and sequencing showed that the △crp△asd C79-13(pcD-asd) balanced lethal system was constructed successfully.Biological characteristics showed that the serotype of△crp △asdC79-13(pcD-asd) was identical to △crp△asdC79-13 and C79-13;the biochemical characteristics of AcrpAasdC79-13(pcD-asd) were basically the same with △crp△asdC79-13 strain,however,it has a significant different with the wild strain C79-13;growth rate was also more slowly;it was sensitive to ampicillin;the median lethal dose reduces the 3.4×10~4 times of C79-13.Western blot analysis showed that the size of the foreign protein was about 27 KDa,when the recombinant strain infeced CEF cells.These results demonstrated that the new recombinant Salmonella pullorum △crp△asdC79-13(pcD-asd),which can present exogenous antigenwas effectively and have great potential be used as an oral live vaccine vector for expressing foreign genes efficiently and safety and stably,was constructed successfully.
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