旋毛虫Nudix水解酶的分子鉴定及其对BALB/c小鼠旋毛虫病的诱导性保护性免疫
Molecular identification of Trichinella spiralis nudix hydrolase and its induced protective immunity against trichinellosis in BALB/c mice.
作者信息
Long Shao Rong, Wang Zhong Quan, Liu Ruo Dan, Liu Li Na, Li Ling Ge, Jiang Peng, Zhang Xi, Zhang Zi Fang, Shi Hai Ning, Cui Jing
机构信息
Department of Parasitology, Medical College of Zhengzhou University, Zhengzhou, 450052, P. R. China.
Department of Immunology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129, USA.
出版信息
Parasit Vectors. 2014 Dec 19;7:600. doi: 10.1186/s13071-014-0600-9.
BACKGROUND
Nudix hydrolases (Nd) is a widespread superfamily, which is found in all classes of organism, hydrolyse a wide range of organic pyrophosphates and has a 'housecleaning' function. The previous study showed that Trichinella spiralis Nd (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with T7 phage-displayed TsNd polypeptides produced protective immunity. The aim of this study was to clone, express and identify the full-length TsNd and to investigate its immune protection against T. spiralis infection.
METHODS
The full-length cDNA sequence of TsNd gene encoding a 46 kDa protein from T. spiralis intestinal infective larvae (IIL) was cloned and identified. The antigenicity of rTsNd was analyzed by Western blot. Transcription and expression of TsNd at T. spiralis different stages were observed by RT-PCR and IFT. The levels of the specific total IgG, IgG1 and IgG2a antibodies to rTsNd were determined by ELISA. The immune protection of rTsNd against T. spiralis infection was investigated.
RESULTS
Sequence and phylogenetic analysis revealed that TsNd had a nudix motif located at 226-244aa, which had high homology and the closest evolutionary status with T. pseudospiralis. The rTsNd was obtained after expression and purification. Western blot analysis showed that anti-rTsNd serum recognized the native TsNd protein in crude antigens of muscle larvae (ML), IIL, adult worms (AW) and newborn larvae (NBL), and ES antigens of ML. Transcription and expression of TsNd gene was observed in all developmental stages of T. spiralis (ML, IIL, AW and NBL), with high level expression in IIL. An immunolocalization analysis identified TsNd in the cuticle, stichocytes and reproductive organs of the parasite. Following immunization, anti-rTsNd IgG levels were increased, and the levels of IgG1 were more significantly higher than that of IgG2a. After a challenge infection with T. spiralis, mice immunized with the rTsNd displayed a 57.7% reduction in adult worms and a 56.9% reduction in muscle larval burden.
CONCLUSIONS
TsNd induced a partial protective immunity in mice and could be considered as a novel candidate vaccine antigen against trichinellosis.
背景
Nudix水解酶（Nd）是一个广泛存在的超家族，存在于所有生物类别中，能水解多种有机焦磷酸，具有“清洁”功能。先前的研究表明，旋毛虫Nd（TsNd）与肠上皮细胞（IECs）结合，用T7噬菌体展示的TsNd多肽对小鼠进行疫苗接种可产生保护性免疫。本研究的目的是克隆、表达和鉴定全长TsNd，并研究其对旋毛虫感染的免疫保护作用。
方法
克隆并鉴定了编码来自旋毛虫肠道感染性幼虫（IIL）的46 kDa蛋白的TsNd基因的全长cDNA序列。通过蛋白质印迹分析rTsNd的抗原性。通过RT-PCR和IFT观察TsNd在旋毛虫不同阶段的转录和表达。通过ELISA测定针对rTsNd的特异性总IgG、IgG1和IgG2a抗体水平。研究rTsNd对旋毛虫感染的免疫保护作用。
结果
序列和系统发育分析表明，TsNd在226-244aa处有一个nudix基序，与伪旋毛虫具有高度同源性和最接近的进化地位。表达和纯化后获得了rTsNd。蛋白质印迹分析表明，抗rTsNd血清可识别肌肉幼虫（ML）、IIL、成虫（AW）和新生幼虫（NBL）的粗抗原以及ML的ES抗原中的天然TsNd蛋白。在旋毛虫的所有发育阶段（ML、IIL、AW和NBL）均观察到TsNd基因的转录和表达，在IIL中表达水平较高。免疫定位分析在寄生虫的角质层、杆状体细胞和生殖器官中鉴定出TsNd。免疫后，抗rTsNd IgG水平升高，IgG1水平比IgG2a水平显著更高。在用旋毛虫进行攻击感染后，用rTsNd免疫的小鼠成虫减少了57.7%，肌肉幼虫负荷减少了56.9%。
结论
TsNd在小鼠中诱导了部分保护性免疫，可被视为抗旋毛虫病的新型候选疫苗抗原。
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