弓形虫GJS株表面抗原1基因的原核细胞表达及其抗原性分析  

Prokaryotic cell expression of the surface antigen 1 gene from GJS strain of Toxoplasma gondii and its antigenicity analysis

作　　者：曹丽艳[1,2] 张德林[1] 张燕丽[1,2] 芦赟[1,2] 蔡志杰[1,2] 王艳华[1] 赵晋军[2] 

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点开放实验室,甘肃省动物寄生虫病重点实验室,兰州730046 [2]甘肃农业大学动物医学院,兰州730070

出　　处：《中国人兽共患病学报》2009年第8期804-809,共6页Chinese Journal of Zoonoses

基　　金：国家科技支撑计划项目(2007BAD40B05);公益性行业(农业)科研专项经费项目资助(No.200803017);甘肃省重大专项科技项目(2GS063-A43-013)资助

摘　　要：目的构建弓形虫GJS株表面抗原1(SAG1)重组表达质粒,研究SAG1蛋白疫苗诱导的保护性免疫作用。方法根据RH株弓形虫SAG1基因序列设计1对引物,利用PCR方法获得SAG1基因,克隆入pMD18-T载体,测序后进行序列分析;重新设计引物将其亚克隆至原核表达载体pET-30a中,在大肠杆菌中经IPTG诱导表达;重组抗原经纯化和复性后免疫小鼠,ELISA法测定其抗体滴度的变化,并用弓形虫速殖子攻击检测免疫保护力。结果与已知的SAG1基因核苷酸序列(S76248)及其编码氨基酸序列的同源性分别为99%、97%;表达的SAG1蛋白以包涵体形式存在,该重组抗原能被羊抗弓形虫阳性血清所识别;经间接ELISA检测,小鼠免疫后产生了较高的抗体;动物保护性实验表明,虽然与对照组相比免疫组小鼠存活时间有一定的延长,但差异无显著性。结论成功构建了弓形虫SAG1重组表达质粒,SAG1蛋白疫苗诱导小鼠的免疫保护力不强。To construct recombinant plasmid DNA encoding I gene from GJS strain of Toxoplasrna gondii （SAG1） and investigate the protective immunity induced by SAG1 protein vaccine. One pair of specific primers based on the sequnces of the surface antigen gene of T. gondii RH strain was designed. SAG1 was obtained through PCR, cloned into PMD-18T vector and sequencing; and was subcloned into the vector PET -30a, its expression was induced by IPTG; Purified and refolded SAG1 （rSAG1） was used to immunize mice and to detect IgG antibodies by ELISA. All mice were challenged with highly virulent tachyzoites to test the protective immunity. Homology analysis showed that the ORF of nucleotide sequences and amino acid sequences of SAG1 shared 99% and 97% identity with those of the published T. gondii SAG1 sequence （S76248）; The expressed product existed in a form of inclusion body and could be recognized by the goat serum against T. gondii; The SAG1 protein vaccine could induce high titer of anti-SAG1 antibodies in immunized mice; The protective trial proved that there was no significant difference between control group and experimental group though the survival time of mice from experimental group had been prolonged. It is evident that the expression plasmids containing the gene fragment enconding SAG1 surface antigen from T gondii have been successfully constructed; hot protective immunity effects of SAG1 protein vaccine to mice was not strong.

关 键 词：弓形虫 表面抗原1 克隆表达 抗原性分析 

分 类 号：R382.5[医药卫生—医学寄生虫学]