微小隐孢子虫分泌蛋白cgd2420的鉴定

Identification of a secreted protein of Cryptosporidium parvum

目的针对微小隐孢子虫一个含信号肽的功能未知蛋白进行细胞定位等分析,为阐明其功能提供实验依据。方法通过生物信息学分析从隐孢子虫基因组寻找含信号肽蛋白,从中选出cgd2420基因编码的蛋白,以此为对象设计抗原多肽并免疫新西兰家兔,获得多克隆抗体;利用膜亲和层析膜纯化方法纯化抗体,利用免疫荧光(IFA)法和ELISA试验检测该蛋白在子孢子和脱囊阶段的亚细胞定位和分泌情况。结果 IFA显示cgd2420蛋白位于子孢子表膜,并在滑行过程分泌形成轨迹。ELISA结果显示,抗cgd2420抗体在隐孢子虫脱囊后的上清中检测到该蛋白(A405值为0.532),是对照样品未脱囊上清(A405值为0.145)的3.65倍(P<0.01)。结论 cgd2420蛋白是一种位于子孢子表膜的分泌蛋白,在卵囊脱囊阶段及子孢子滑行过程中分泌,这些特征表明该蛋白可能是潜在的分泌抗原,值得进一步探究其在子孢子入侵时期的作用,以及作为检测隐孢子虫感染的候选靶抗原的可行性。

Objective This study focused on identifying and characterizing a signal peptide-containing protein from the protozoan parasite Cryptosporidium parvum,aiming to provide primary experimental data towards understanding its function. Methods A number of proteins containing an N-terminal signal peptide were identified from the genome of C. parvum by bioinformatic analysis. Among them, a protein encoded by cgd2420 gene annotated as "Signal peptide containing protein" was selected for designing a short peptide antigen and immunizing rabbits to generate antiserum. Specific antibody was purified by a membrane-based affinity chromatography for detecting cgd2420 protein in the sporozoites during and after excystation by immunofluorescence assay(IFA) and ELISA. Results cgd2420 protein was localized to the surface of sporozoites and could be discharged from the sporozoites to form gliding tracks on glass slides as observed by IFA. ELISA results showed that the mean A405 value of supernant from post excystation group were 0.532,a 3.6 fold increase over the control group(A405 value=0.145) Student’s t-test showed that both groups are statistically different(P<0.01). Conclusion cgd2420 protein is a surface protein that can be secreted from sporozoites during the excystation and invasion processes. These features suggest that this protein is worth to be explored further as a potential antigen target for the detection of Cryptosporidium infection.