粪类圆线虫虾青素金属蛋白酶基因的克隆、表达及定位分析

Identification and expression of Ss-ast-1 astacin metalloproteinase encoding gene of Strongyloides stercoralis

虾青素金属蛋白酶是一种广泛存在于多种动物体内的金属蛋白酶,研究表明该蛋白酶在寄生线虫的入侵和生长发育过程中发挥重要作用。但目前对粪类圆线虫虾青素金属蛋白酶(Ss-AST-1)还所知甚少,研究旨在对该酶编码基因(Ss-ast-1)进行鉴定和表达分析,为进一步研究其功能奠定基础。利用生物信息学方法对Ss-ast-1基因进行序列分析,通过转录组数据分析Ss-ast-1在各个发育阶段的转录水平,转基因技术进行虫体内表达定位分析。通过体外原核表达重组蛋白,Western blot分析其抗原性。采用免疫荧光组织化学方法对Ss-AST-1在粪类圆线虫幼虫体内的定位进行研究。结果显示:Ss-ast-1基因全长1905 bp,编码634个氨基酸;转录组分析该基因在iL3期的表达水平最高。定位分析表明该蛋白在虫卵细胞的细胞核和细胞质均有表达;体外表达重组蛋白的大小约67.5 kDa,Western blot分析表明多克隆抗体可以较好的识别全虫蛋白中的Ss-AST-1。免疫荧光结果显示Ss-AST-1在幼虫体内表达并分布幼虫的头部和体壁。Ss-ast-1属于虾青素金属蛋白酶家族,其可能在寄生虫入侵过程中发挥作用,为进一步探究粪类圆线虫蛋白功能奠定了良好的基础。

Astacin metalloprotease is a metalloprotease widely found in various animals.Studies have shown that this protease plays an important role in growth of parasitic nematodes and the process of invasion of the host.However,little is known about their functions in parasitic nematode Strongyloides stercoralis(S.stercoralis).In this study,bioinformatics methods were used to analyze the transcriptional profiles of Ss-ast-1 at various developmental stages in S.stercoralis and analyze the transcriptional localization in vivo by transgenesis.The recombinant protein was expressed in E.coli in vitro,and its antigenicity was analyzed by Western blot.The localization of Ss-AST-1 in S.stercoralis was studied by immunofluorescence histochemistry.The Ss-ast-1 gene was 1905 bp in length and encoded 634 amino acids.Transcriptome analysis revealed that Ss-ast-1 was transcribed throughout the development with the highest transcriptional level at iL3 larval stage.Localization analysis showed that Ss-ast-1 was expressed in the nucleus and cytoplasm of egg cells.The size of the recombinant protein Ss-AST-1 expressed in E.coli was 67.5 kDa.Western blot analysis showed that anti-Ss-AST-1 polyclonal antibodies recognized the endogenous protein from whole worm proteins.Immunofluorescence results showed that Ss-AST-1 was expressed in the head and body wall of the larvae of S.stercoralis.Ss-ast-1 belongs to the family of astacin metalloprotease,which may play a role in the process of parasite invasion.This study lays the foundation for further exploring the function of Ss-AST-1 protein in S.stercoralis.