重组甲鱼载脂蛋白B的研制及其阻断STSSSV感染的活性初探

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, 显微镜下观察, 结果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.

Preparation and STSSSV infection blocking assay of recombinant PApoB

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 soft- shelled turtle (Pelodiscus sinensis) systemic septicemia spherical virus (STSSSV) Primers were designed referring to PApoB gene to amplify a 1 277 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 PApoB- supplemented 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.