作　　者： ; ; ; ; ;

机构地区： 华南农业大学兽医学院

出　　处： 《中国兽医科学》 2015年第1期1-7,共7页

摘　　要： 为研究弓形虫profilin(prf)蛋白基因的遗传变异规律和表达产物的反应原性,以9个来源于不同宿主和地方的弓形虫虫株基因组DNA为模板,利用PCR方法扩增该基因并测序。将测序结果与从ToxoDB网站下载的4株弓形虫序列一起进行比对分析,用最大简约法绘制系统发育树。再以弓形虫RH株虫体总RNA为模板,使用RT-PCR方法扩增prf基因片段,并将其克隆到原核表达载体pET-30a(+)中,转化入表达菌BL21(DE3)后,使用IPTG进行诱导表达,应用Western-blot对重组蛋白的反应原性进行分析。同时,将弓形虫RH株prf基因编码区序列翻译成氨基酸序列,利用生物信息学软件对其亲疏水性、跨膜结构域、二级结构及B细胞抗原表位进行分析。结果显示,9个弓形虫虫株的prf基因长度均为3 552bp,一致性大于99.5%。系统发育树分析表明,弓形虫虫株的聚类没有规律。SDS-PAGE和Western-blot分析结果显示,Profilin蛋白的分子质量约为30ku,且反应原性较好。蛋白结构预测结果显示,该蛋白含有4个亲水区,无跨膜区,7个α螺旋,8个β折叠,4个β转角,2个无规则卷曲和7个线性B细胞抗原表位。本研究结果表明,弓形虫prf基因可作为抗弓形虫疫苗候选分子。 The objectives of the present study were to examine sequence variation and reactionogenicity of Toxoplasma gondii profilin(prf)protein gene.T.gondii profilin protein gene was amplified from 9 T.gondii strains from different hosts and geographical locations by PCR and sequenced.Then the phylogenetic relationships among these T.gondii strains were reconstructed based on the obtained sequences and the corresponding sequences available in ToxoDB by maximum parasimony(MP)method.To study the reactionogenicity of T.gondii profilin protein,the gene coding for profilin was amplified by RT-PCR,and then the product was linked into prokaryotic expression vector pET-30a(+)and transformed into Escherichia coli BL21(DE3)strain.The profilin protein was expressed by inducing with IPTG and its reactionogenicity was analyzed by Western-blot.Meanwhile,the hydrophobicity,transmembrane domain,secondary structure and the B cell epitopes were predicted by bioinformatics softwares using the amino acid sequence translated fromT.gondii profilin gene sequences.In result,the length of the profilin protein gene from 9 T.gondii isolates was all 3 552 bp,with an identity of 99.5%.Phylogenetic analysis showed that the profilin protein gene sequences could not differentiate all genotypes of T.gondii.SDS-PAGE and Western-blot analyses showed that the profilin protein was about 30 ku in molecular weight,and possessed good reactionogenicity.Bioinformatics analysis revealed that the profilin protein contained 4hydrophobicity regions,7alpha regions,8beta sheets,4beta turns,2random coils and 7Bcell epitopes.These results suggested that T.gondii profilin could be one of promising vaccine candidates against T.gondii infection,which provide useful information for further development of new T.gondii DNA vaccines or peptide vaccines.

关 键 词： 弓形虫 蛋白基因 遗传变异 序列分析 原核表达 细胞抗原表位

领　　域： [农业科学] [农业科学] [农业科学]