重组甲鱼载脂蛋白B的研制及其阻断STSSSV感染的活性初探  

Preparation and STSSSV infection blocking assay of recombinant PApoB

作　　者：许丽华 顾叶华[1] 陈梅娟[1] 李登峰[1] XU Lihua;GU Yehua;CHEN Meijuan;LI Dengfeng(Key Laboratory of Applied Technology of Marine Biology of Ministry of Education,Ningbo University,Ningbo 315832,China)

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室

出　　处：《宁波大学学报（理工版）》2020年第2期7-12,共6页Journal of Ningbo University：Natural Science and Engineering Edition

基　　金：国家重点研发计划(2018YFA0903000);浙江省公益性技术应用研究计划(2011C32004);宁波市农业攻关项目(2011C10004)

摘　　要：为了制备甲鱼(Pelodiscus sinensis)载脂蛋白B(Pelodiscus sinensis Apolipoprotein B,PApoB)基因的原核表达产物,并探索其是否具有阻断甲鱼系统性败血症球状病毒(Soft-shelled Turtle Systemic Septicemia Spherical Virus,STSSSV)感染的功能,为最终控制和防治甲鱼系统性败血症疾病奠定基础,构建了PApoB的原核表达系统,纯化了重组PApoB,并将其用于阻断STSSSV感染的分析:根据甲鱼PApoB的基因序列,设计引物,扩增其第9932-11209bp(3294-3696 aa)区段,将其连接至表达载体pET-28a(+)中,转化大肠杆菌Rosetta株,经硫代半乳糖苷(IPTG)诱导表达,并用亲和层析法纯化获得了重组PApoB;将FITC标记的STSSSV与PApoB孵育后对甲鱼细胞进行攻毒,显微镜下PApoB添加组的病毒荧光信号明显低于非添加组,表明该重组蛋白可以阻断STSSSV的感染.The current study was aimed to prepare the recombinant protein of Pelodiscus sinensis Apolipoprotein B(PApoB)and examine whether the recombinant PApoB has the function of blocking the infection of softshelled turtle(Pelodiscus sinensis)systemic septicemia spherical virus(STSSSV)Primers were designed referring to PApoB gene to amplify a 1277 bp fragment.The fragment was ligated into the expression vector pET-28a(+).Subsequently,the recombinant plasmid was transformed into Escherichia coli Rosetta and was induced by the thiogalactoside(IPTG).PApoB was purified by affinity chromatography.Following the incubation of FITC-labeled STSSSV and recombinant PApoB,the soft-shelled turtle cells were challenged and observed under fluorescence microscope.Results showed that the fluorescence signal of the virus in the PApoBsupplemented groups was significantly lower than that of the non-PApoB-addition groups.The results indicate that the recombinant protein was effective to inhibit the infection of STSSSV.

关 键 词：甲鱼 载脂蛋白B STSSSV 原核表达 感染阻断 

分 类 号：S948[农业科学—水产养殖]