携带NDV HN 基因的减毒鼠伤寒沙门菌Ⅲ型分泌系统构建及其生物学特性分析  

Construction and Biological Characterization of an Attenuated Salmonella typhimurium TypeⅢSecretion System Carrying NDV HN Gene

作　　者：宋敏杰 周小双 石胜丽 朱文文 田文静 程相朝 郁川 SONG Minjie;ZHOU Xiaoshuang;SHI Shengli;ZHU Wenwen;TIAN Wenjing;CHENG Xiangchao;YU Chuan(Pet&Human Health Engineering Technology Center,Luoyang Vocational and Technical College,Luoyang 471900,China;College of Animal Science and Technology,Henan University of Science and Technology/Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control,Luoyang 471023,China;Xinxiang Animal Health Supervision Institute,Xinxiang 453000,China)

机构地区：[1]洛阳职业技术学院宠物与人类健康工程技术中心,河南洛阳471900 [2]河南科技大学动物科技学院/洛阳市活载体生物材料与动物疫病防控重点实验室,河南洛阳471023 [3]新乡市动物卫生监督所,河南新乡453000

出　　处：《河南农业科学》2021年第10期116-123,共8页Journal of Henan Agricultural Sciences

基　　金：河南省科技攻关项目(152102110078)。

摘　　要：为构建能稳定携带新城疫病毒(Newcastle disease virus,NDV)血凝素-神经氨酸酶蛋白(Hemagglutinin‑neuraminidase protein,HN)基因的减毒鼠伤寒沙门菌Ⅲ型分泌系统,从pMD18-T-HN中扩增出NDV HN基因,并将其克隆入载体pYA3493-sopENt100,构建重组载体pYA3493-sopENt100-HN,然后将其电转化至减毒鼠伤寒沙门菌ΔcrpΔasd SL1344,并对其重组菌的生长特性、遗传稳定性、表达特性和毒力特性等进行分析。结果显示,携带NDV HN基因的重组减毒鼠伤寒沙门菌ΔcrpΔasd SL1344(pYA3493-sopENt100-HN)构建成功;重组菌ΔcrpΔasd SL1344(pYA3493-sopENt100-HN)的生长趋势与互补菌ΔcrpΔasd SL1344(pYA3493-sopENt100)和缺失菌ΔcrpΔasd SL1344相似,与亲本菌株SL1344有明显差异;重组菌能够稳定表达HN基因;减毒鼠伤寒沙门菌Ⅲ型分泌系统能够有效递呈HN基因,并诱导其在鸡胚成纤维细胞(Chicken embryo fibroblast,CEF)内表达;重组菌的LD_(50)与互补菌和缺失菌没有明显差异,与亲本菌株SL1344差异较大。以上结果表明,成功构建了携带NDV HN基因的新型减毒鼠伤寒沙门菌Ⅲ型分泌系统ΔcrpΔasd SL1344(pYA3493-sopENt100-HN),能够有效递呈NDV HN基因并使其高效表达,且安全性高,遗传稳定。In order to construct a recombinant attenuated Salmonella typhimurium typeⅢsecretion system which carries the hemagglutinin‑neuraminidase(HN)protein gene of Newcastle disease virus(NDV),the HN gene was amplified from the plasmind pMD18‑T‑HN and cloned into the vector pYA3493‑sopENt100.Then the recombinant vector pYA3493‑sopENt100‑HN was transformed into attenuated Salmonella typhimurium strainΔcrpΔasd SL1344 by electroporation,and the growth characteristics,genetic stability,expression characteristics and virulence of the recombinant strainΔcrpΔasd SL1344(pYA3493‑sopENt100‑HN)were analyzed.The identification results of PCR and enzyme digestion showed that the recombinant attenuated Salmonella typhimurium strainΔcrpΔasd SL1344(pYA3493‑sopENt100‑HN)was successfully constructed.The growth curve of the recombinant strain was similar to that of the complementary strainΔcrpΔasd SL1344(pYA3493‑sopENt100)and the deletion strainΔcrpΔasd SL1344,which were significantly different from their parent strain SL1344.The HN gene was stably inherited in the recombinant strain.The HN protein was presented by the attenuated Salmonella typhimuriumⅢtype secretion system and expressed in chicken embryo fibroblast(CEF).The LD50 of the recombinant strain was not significantly different from the complementary strain and the deletion strain,but significantly different from the parent strain SL1344.The above results indicated that the recombinant attenuated Salmonella typhimuriumⅢsecretion system carrying the NDV HN gene was successfully constructed,which had high safety,genetic stability,and expression efficacy of the NDV HN gene.

关 键 词：新城疫病毒 HN 基因 Ⅲ型分泌系统 减毒沙门菌 生物学特性 

分 类 号：S858.31[农业科学—临床兽医学]