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
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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.
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.
引文
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[9] Feng J,Zhan B,Liu Y,et al.Molecular cloning and characterization of Ac-MTP-2,an astacin-like metalloprotease released by adult Ancylostoma caninum[J].Mol Biochem Parasitol,2007,152(2):132-8.
[10] Jones BF,Hotez PJ.Molecular cloning and characterization of Ac-mep-1,a developmentally regulated gut luminal metalloendopeptidase from adult Ancylostoma caninum hookworms[J].Mol Biochem Parasitol,2002,119(1):107-16.
[11] Park JO,Pan J,Mohrlen F,et al.Characterization of the astacin family of metalloproteases in C.elegans[J].BMC Dev Biol,2010,28(10):14.
[12] Podbilewicz B.ADM-1,a protein with metalloprotease- and disintegrin-like domains,is expressed in syncytial organs,sperm,and sheath cells of sensory organs in Caenorhabditis elegans[J].Mol Biol Cell,1996,7(12):1877-93.
[13] Hill DE,Gamble HR,Rhoads ML,et al.Trichuris suis:a zinc metalloprotease from culture fluids of adult parasites[J].Exp Parasitol,1993,77(2):170-8.
[14] 梁浩昆,沈浩贤,李烈,等.广州管圆线虫对实验动物致病作用的实验研究[J].广州医学院学报,1984(1):38-47,117.
[15] 朱天成,丁步兰,罗肖萍.广州管圆线虫(Angiostongylus cantonensis)在大白鼠体内的移行、发育及其引起的病理损害的实验观察[J].广州医学院学报,1983(4):8-28.
[2] Lepage T,Ghiglione C,Gache C.Spatial and temporal expression pattern during sea urchin embryogenesis of a gene coding for a protease homologous to the human protein BMP-1 and to the product of the Drosophila dorsal-ventral patterning gene tolloid[J].Development,1992,114(1):147-63.
[3] Stepek G,McCormack G,Winter AD,et al.A highly conserved,inhibitable astacin metalloprotease from Teladorsagia circumcincta is required for cuticle formation and nematode development[J].Int J Parasitol,2015,45(5):345-55.
[4] Feng J,Zhan B,Liu Y,et al.Molecular cloning and characterization of Ac-MTP-2,an astacin-like metalloprotease released by adult Ancylostoma caninum[J].Mol Biochem Parasitol,2007,152(2):132-8.
[5] Jolodar A,Fischer P,Büttner DW,et al.Onchocerca volvulus:expression and immunolocalization of a nematode cathepsin D-like lysosomal aspartic protease[J].Exp Parasitol,2004,107(3-4):145-56.
[6] Davis MW,Birnie AJ,Chan AC,et al.A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans[J].Development,2004,131(23):6001-8.
[7] Jones BF,Hotez PJ.Molecular cloning and characterization of Ac-mep-1,a developmentally regulated gut luminal metalloendopeptidase from adult Ancylostoma caninum hookworms[J].Mol Biochem Parasitol,2002,119(1):107-16.
[8] McKerrow JH,Brindley P,Brown M,et al.Strongyloides stercoralis:identification of a protease that facilitates penetration of skin by the infective larvae[J].Exp Parasitol,1990,70(2):134-43.
[9] Feng J,Zhan B,Liu Y,et al.Molecular cloning and characterization of Ac-MTP-2,an astacin-like metalloprotease released by adult Ancylostoma caninum[J].Mol Biochem Parasitol,2007,152(2):132-8.
[10] Jones BF,Hotez PJ.Molecular cloning and characterization of Ac-mep-1,a developmentally regulated gut luminal metalloendopeptidase from adult Ancylostoma caninum hookworms[J].Mol Biochem Parasitol,2002,119(1):107-16.
[11] Park JO,Pan J,Mohrlen F,et al.Characterization of the astacin family of metalloproteases in C.elegans[J].BMC Dev Biol,2010,28(10):14.
[12] Podbilewicz B.ADM-1,a protein with metalloprotease- and disintegrin-like domains,is expressed in syncytial organs,sperm,and sheath cells of sensory organs in Caenorhabditis elegans[J].Mol Biol Cell,1996,7(12):1877-93.
[13] Hill DE,Gamble HR,Rhoads ML,et al.Trichuris suis:a zinc metalloprotease from culture fluids of adult parasites[J].Exp Parasitol,1993,77(2):170-8.
[14] 梁浩昆,沈浩贤,李烈,等.广州管圆线虫对实验动物致病作用的实验研究[J].广州医学院学报,1984(1):38-47,117.
[15] 朱天成,丁步兰,罗肖萍.广州管圆线虫(Angiostongylus cantonensis)在大白鼠体内的移行、发育及其引起的病理损害的实验观察[J].广州医学院学报,1983(4):8-28.