广州管圆线虫虾红素金属蛋白酶基因的克隆、表达及免疫组织定位分析  

Molecular cloning, expression, and immunohistochemical localization of astacin-like metalloproteases (Ac-ALMPs) from Angiostrongylus cantonensis

作　　者：余良 谷麦 曹斌斌 罗大民[1] YU Liang;GU Mai;CAO Bin-bin;LUO Da-min(College of Life Sciences,Xiamen University ,Xiamen ,Fujian,China 361102)

机构地区：[1]厦门大学生命科学学院,福建厦门361102

出　　处：《中国病原生物学杂志》2019年第4期426-432,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81171595)

摘　　要：目的通过对广州管圆线虫虾红素金属蛋白酶基因的克隆、表达及免疫组织定位分析,探讨其在虫体中的分布及功能。方法根据虾红素金属蛋白酶EST序列设计引物进行RACE克隆和进化树分析;制备Ac-ALMP多抗血清,对广州管圆线虫L1,感染期L3和成虫进行免疫组织定位,通过Real-time PCR进行Ac-ALMP定量分析。结果获得广州管圆线虫虾红素金属蛋白酶全基因序列,并将其命名为Ac-ALMP(Astacin-like metalloprotease)。进化树分析Ac-ALMP基因的核酸序列与Dictyocaulus viviparous同源性较高。免疫荧光定位分析Ac-ALMP在广州管圆线虫的各期中主要分布于消化道(肠道与食道)。定量分析显示Ac-ALMP在各期中均有表达,其中L1相对表达量较高,L3次之。结论成功对Ac-ALMP蛋白进行了原核及真核表达,定量分析Ac-ALMP在虫体的肠道、角质层、精巢、体壁等均有分布,推测Ac-ALMP在广州管圆线虫寄生过程中起重要作用,可为广州管圆线虫病的防治及疫苗开发提供参考。Objective To investigate the distribution and function of astacin-like metalloproteases of Angiostrongylus cantonensis(Ac-ALMPs)via cloning,expression,and immunohistochemical localization.Methods A full-length or partial coding sequence of an Ac-ALMP was amplified with gene-specific primers using rapid amplification of cDNA ends(RACE),and then phylogenetic analysis was performed.Ac-ALMPs were immunohistochemically localized using polyclonal antisera and quantitatively analyzed in L1,L3 and the adult stage using real-time PCR.Results The complete genetic sequence of a metalloproteinase from A.cantonensis was obtained and designated Ac-ALMP.Phylogenetic tree analysis indicated that the nucleotide sequence of the Ac-ALMP gene was most similar to Dictyocaulus viviparous.Immunofluorescence localization indicated that Ac-ALMP was mainly distributed in the digestive tract(intestine and esophagus)of A.cantonensis.Quantitative results indicated that Ac-ALMP was expressed in all stages,and the relative expression was highest in L1,followed by L3,suggesting that Ac-ALMP played an important role in parasitism.Conclusion In this study,prokaryotic and eukaryotic expression of the Ac-ALMP protein was used to provide an antigen for diagnosis of A.cantonensis.This study speculated on the function of Ac-ALMP in A.cantonensis by studying the localization of Ac-ALMP in the parasite,and findings have provided a reference for the prevention and control of A.cantonensis and the development of vaccines.

关 键 词：广州管圆线虫 虾红素金属蛋白酶 多克隆抗血清 免疫定位 

分 类 号：R383.1[医药卫生—医学寄生虫学]