微小隐孢子虫Ⅰ型跨膜蛋白Cgd7＿4560的亚细胞定位及其对HCT-8细胞的黏附特性
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摘要
型跨膜蛋白在顶复门寄生虫入侵宿主细胞过程中发挥重要作用。试验对微小隐孢子虫Ⅰ型跨膜蛋白Cgd7_4560进行生物信息学分析,选取抗原性强的序列,采用PCR方法扩增出微小隐孢子虫DNA Cgd7_4560部分基因。将该片段分别连接到p ET-28a和p GEX-4T-1载体,构建重组表达质粒p ET-28a-Cgd7_4560和p GEX-Cgd7_4560,并将其转化到大肠埃希氏杆菌BL21 (Condon plus),IPTG诱导表达获得重组蛋白。SDSPAGE和Western blot分析,His标签重组蛋白为31 ku,GST标签重组蛋白为53 ku。用His标签重组蛋白免疫家兔,间接ELISA方法检测血清抗体效价达到1∶16 000,成功制备多克隆抗体。采用Western blot方法验证该抗体能够特异性的识别虫体天然蛋白,间接免疫荧光分析该蛋白在子孢子头端,间接ELISA结果表明GSTCgd7_4560能与HCT-8细胞黏附,为其功能研究奠定了基础。
TypeⅠtransmembrane proteins play an important role during the invasion of phylum Apicomplexa parasites. Bioinformatics analysis of typeⅠtransmembrane proteins Cgd7_4560 of Cryptosporidium parvum was carried out to select the highly antigenic part of Cgd7_4560. The PCR fragments were inserted to the vectors of pET-28 a and pGEX-4 T-1,respectively,to construct the recombinant expression plasmids pET-Cgd7_4560 and pGEX-Cgd7_4560,and they were transformed into Escherichia coli BL21( Condon plus). IPTG was used to induce the expression of recombinant protein. The recombinant proteins were analyzed by SDS-PAGE and Western blot,and a His-tagged31 ku recombinant protein and a 53 ku GST-tagged recombinant protein were identified. Rabbit was immunized with His-tagged recombinant protein,and the titer of antibody in serum was 1 ∶ 16 000 by indirect ELISA. Native Cgd7_4560 protein could be recognized at 340 ku by Western blot. Indirect immunofluorescence analysis of the protein suggested that it is localized in the apical end of sporozoites. Cell binding ELISA results showed that recombinant protein can attach to HCT-8 cells in dose-dependent manner. These findings lay foundation for future functional research of the Cgd7 _4560 in Cryptosporidium parvum.
TypeⅠtransmembrane proteins play an important role during the invasion of phylum Apicomplexa parasites. Bioinformatics analysis of typeⅠtransmembrane proteins Cgd7_4560 of Cryptosporidium parvum was carried out to select the highly antigenic part of Cgd7_4560. The PCR fragments were inserted to the vectors of pET-28 a and pGEX-4 T-1,respectively,to construct the recombinant expression plasmids pET-Cgd7_4560 and pGEX-Cgd7_4560,and they were transformed into Escherichia coli BL21( Condon plus). IPTG was used to induce the expression of recombinant protein. The recombinant proteins were analyzed by SDS-PAGE and Western blot,and a His-tagged31 ku recombinant protein and a 53 ku GST-tagged recombinant protein were identified. Rabbit was immunized with His-tagged recombinant protein,and the titer of antibody in serum was 1 ∶ 16 000 by indirect ELISA. Native Cgd7_4560 protein could be recognized at 340 ku by Western blot. Indirect immunofluorescence analysis of the protein suggested that it is localized in the apical end of sporozoites. Cell binding ELISA results showed that recombinant protein can attach to HCT-8 cells in dose-dependent manner. These findings lay foundation for future functional research of the Cgd7 _4560 in Cryptosporidium parvum.
引文
[1] O'Donoghue P J. Cryptosporidium and cryptosporidiosis in man and animals[J].Int J Parasitol,1995,25(2):139-195.
[2]王科杰,邵清延,张天宇,等.微小隐孢子虫TSP3蛋白的定位分析[J].吉林农业大学学报,2017,39(2):199-203.
[3] Graaf D C,Vanopdenbosch E,Ortega-Mora L M,et al. A review of the importance of cryptosporidiosis in farm animals[J].Int J Parasitol,1999,29(8):1269-1287.
[4] Cabada M M,White A C Jr. Treatment of cryptosporidiosis:do we know what we think we know?[J]. Curr Opin Infect Dis,2010,23(5):494-499.
[5] Carruthers V B,Sibley L D. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts[J]. Eur J Cell Biol,1997,73(2):114-123.
[6] Soldati D,Dubremetz J F,Lebrun M. Microneme proteins:structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii[J].Int J Parasitol,2001,31(12):1293-1302.
[7] Tomley F M,Soldati D S. Mix and match modules:structure and function of microneme proteins in apicomplexan parasites[J]. Trends Parasitol,2001,17(2):81-88.
[8] Barnes D A,Bonnin A,Huang J X,et al. A novel multi-domain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion[J]. Mol Biochem Parasitol,1998,30; 96(1-2):93-110.
[9] Putignani L, Possenti A, Cherchi S, et al. The thrombospondin-related protein CpMIC1(CpTSP8)belongs to the repertoire of micronemal proteins of Cryptosporidium parvum[J]. Mol Biochem Parasitol,2008,157(1):98-101.
[10] Spano F,Putignani L,Naitza S,et al. A. Molecular cloning and expression analysis of a Cryptosporidium parvum gene encoding a new member of the thrombospondin family[J]. Mol Biochem Parasitol,1998,92(1):147-162.
[11] Arrowood M J,Sterling C R. Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients[J]. J Parasitol,1987,73(2):314-319.
[12] Saini D K,Pant N,Das T K,et al. Cloning,overexpression,purification, and matrix-assisted refolding of Dev S(Rv3132c)histidine protein kinase of Mycobacterium tuberculosis[J]. Protein Expr Purif,2002,25(1):203-208.
[13] Wan K L,Carruthers V B,Sibley L D,et al. Molecular characterisation of an expressed sequence tag locus of Toxoplasma gondii encoding the micronemal protein MIC2[J]. Mol Biochem Parasitol,1997,84(2):203-214.
[14] Ludington J G,Ward H D. The Cryptosporidium parvum Ctype lectin Cp Clec mediates infection of intestinal epithelial cells via interactions with sulfated proteoglycans[J]. Infect Immun,2016,84(5):1593-1602.
[15] Bhat N,Joe A,Pereira Perrin M. Cryptosporidium p30,agalactose/N-acetylgalactosamine-specific lectin,mediates infection in vitro[J]. J Biol Chem,2007,282(48):34877-34887.
[2]王科杰,邵清延,张天宇,等.微小隐孢子虫TSP3蛋白的定位分析[J].吉林农业大学学报,2017,39(2):199-203.
[3] Graaf D C,Vanopdenbosch E,Ortega-Mora L M,et al. A review of the importance of cryptosporidiosis in farm animals[J].Int J Parasitol,1999,29(8):1269-1287.
[4] Cabada M M,White A C Jr. Treatment of cryptosporidiosis:do we know what we think we know?[J]. Curr Opin Infect Dis,2010,23(5):494-499.
[5] Carruthers V B,Sibley L D. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts[J]. Eur J Cell Biol,1997,73(2):114-123.
[6] Soldati D,Dubremetz J F,Lebrun M. Microneme proteins:structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii[J].Int J Parasitol,2001,31(12):1293-1302.
[7] Tomley F M,Soldati D S. Mix and match modules:structure and function of microneme proteins in apicomplexan parasites[J]. Trends Parasitol,2001,17(2):81-88.
[8] Barnes D A,Bonnin A,Huang J X,et al. A novel multi-domain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion[J]. Mol Biochem Parasitol,1998,30; 96(1-2):93-110.
[9] Putignani L, Possenti A, Cherchi S, et al. The thrombospondin-related protein CpMIC1(CpTSP8)belongs to the repertoire of micronemal proteins of Cryptosporidium parvum[J]. Mol Biochem Parasitol,2008,157(1):98-101.
[10] Spano F,Putignani L,Naitza S,et al. A. Molecular cloning and expression analysis of a Cryptosporidium parvum gene encoding a new member of the thrombospondin family[J]. Mol Biochem Parasitol,1998,92(1):147-162.
[11] Arrowood M J,Sterling C R. Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients[J]. J Parasitol,1987,73(2):314-319.
[12] Saini D K,Pant N,Das T K,et al. Cloning,overexpression,purification, and matrix-assisted refolding of Dev S(Rv3132c)histidine protein kinase of Mycobacterium tuberculosis[J]. Protein Expr Purif,2002,25(1):203-208.
[13] Wan K L,Carruthers V B,Sibley L D,et al. Molecular characterisation of an expressed sequence tag locus of Toxoplasma gondii encoding the micronemal protein MIC2[J]. Mol Biochem Parasitol,1997,84(2):203-214.
[14] Ludington J G,Ward H D. The Cryptosporidium parvum Ctype lectin Cp Clec mediates infection of intestinal epithelial cells via interactions with sulfated proteoglycans[J]. Infect Immun,2016,84(5):1593-1602.
[15] Bhat N,Joe A,Pereira Perrin M. Cryptosporidium p30,agalactose/N-acetylgalactosamine-specific lectin,mediates infection in vitro[J]. J Biol Chem,2007,282(48):34877-34887.