Cloning and expression analysis of a functional fragment of apoptosis-inducing factor (AIF) gene in Schistosoma japonicum
LU Kan1,2,HAN Hong-xiao2,HONG Yang2,MA Qian-qian2, LIU Yan-tao2,HAN Qian2,MA Shuai2, FU Zhi-qiang2,LIN Jiao-jiao2,3
1.College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, China; 2.Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science / Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai 200241, China; 3.Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
Abstract:We cloned and expressed a functional fragment of apoptosis-inducing factor (AIF) gene in Schistosoma japonicum and further analyzed its biological characteristics. PCR technique was employed to amplify the functional fragment of SjAIF by employing a cDNA of 14 d (day) schistosomula as template. The fragment of AIF was subcloned into a pET28a(+) vector and the recombinant plasmid was transformed into competent E.coil/BL21 for producing recombinant protein. The expression level of SjAIF was determined at several different development stages of schistosomula and adult worms by using real-time RT-PCR. The recombinant protein was purified and then its antigenicity was accessed by Western blotting and ELISA. The distribution of the protein in Schistosoma japonicum was analyzed by immunolocalization. Real-time PCR analysis revealed that the expression of SjAIF was lower in 7 d, 14 d and 21 d than that in other stages. Western-blotting showed that the recombinant had good immunogenicity. The vaccinated group showed a good ability to induce IgG as examined by ELISA. Immunolocalization analysis revealed that the SjAIF was mainly distributed in tegument and parenchyma. The fragment of SjAIF was obtained and its molecular characterizations were preliminarily investigated. This study provided an important basis for further investigation of the biological characteristics and mechanism of the protein in Schistosoma japonicum
陆看,韩宏晓,洪炀,马茜茜,刘艳涛,韩倩,马帅,傅志强,林矫矫. 日本血吸虫凋亡诱导因子SjAIF功能区片段的克隆及表达分析[J]. 中国人兽共患病学报, 2015, 31(2): 102-108.
LU Kan,HAN Hong-xiao,HONG Yang,MA Qian-qian, LIU Yan-tao,HAN Qian,MA Shuai, FU Zhi-qiang,LIN Jiao-jiao. Cloning and expression analysis of a functional fragment of apoptosis-inducing factor (AIF) gene in Schistosoma japonicum. Chinese Journal of Zoonoses, 2015, 31(2): 102-108.
[1]Zhang SY, Jiang SF. Development plan of the schistosomiasis vaccines[J]. Foreign Med Sci Parasit Dis, 2004, 31(2): 66-68. (in Chinese) 张述义, 蒋守富. 血吸虫病疫苗开发行动计划[J].国外医学: 寄生虫病分册, 2004, 31(2): 66-68. [2]Liang YS, Li HJ, Dai JR, et al. Studies on resistance of Schistosoma to praziquantel XIII resistance of Schistosoma japonicum to praziquantel is experimentally induced in laboratory[J]. Chin J Schisto Ctrl, 2011, 23(6): 605-610. (in Chinese) 梁幼生, 李洪军, 戴建荣, 等. 血吸虫对吡喹酮抗药性的研究XIII日本血吸虫吡喹酮抗药性的实验诱导[J]. 中国血吸虫病防治杂志, 2011, 23(6): 605-610. [3]Cioli D. Chemotherapy of schistosomiasis: an update[J]. Parasitol Today, 1998, 14(10): 418-422. DOI: 10.1016/S0169-4758(98)01323-4 [4]Peng JB, Hong Y, Gobert GN, et al. Apoptosis governs the elimination of Schistosoma japonicum from the non-permissive host Microtus fortis[J]. PLoS One, 2011, 6(6): e21109. DOI: 10.1371/journal.pone.0021109 [5]Guo XY, Hong Y, Han HX, et al. Observation of apoptosis in 14 days-old Schistosomulum of Schistosoma japonicum[J]. Chin Vet Sci, 2014, 44(06): 558-562. (in Chinese) 郭小勇, 洪炀, 韩宏晓, 等. 日本血吸虫14d童虫凋亡现象的观察[J]. 中国兽医科学, 2014, 44(06): 558-562. [6]Han HX, Peng JB, Gobert GN, et al. Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum[J]. Parasitol Int, 2013, 62(2): 100-108. DOI: 10.1016/j.parint.2012.09.008 [7]Li H, He YY, Lin JJ, et al. The observation for the phenomenon of Microtus fortis aganisting Schistosoma japonicum[J]. Chin J Vet Parasitol, 2000, 8(2): 12-15. (in Chinese) 李浩, 何艳燕, 林矫矫, 等. 东方田鼠抗日本血吸虫病现象的观察[J]. 中国兽医寄生虫病杂志, 2000, 8(2): 12-15. [8]Peng JB, Han HX, Gobert GN, et al. Differential gene expression in Schistosoma japonicum Schistosomula from Wistar rats and BALB/c mice[J]. Parasite Vector, 2011, 4(1): 155-166. DOI: 10.1186/1756-3305-4-155 [9]Beckmann S, Grevelding CG. Imatinib has a fatal impact on morphology, pairing stability and survival of adult Schistosoma mansoni in vitro[J]. Int J Parasitol, 2010, 40(5): 521-526. DOI: 10.1016/j.ijpara.2010.01.007 [10]Wang CZ, Cao C, Ma QJ. The regulation of apoptosis by apoptosis-inducing factor[J]. Chem Life, 2005, 25(6): 454-456. (in Chinese) 王昌正, 曹诚, 马清钧. 凋亡诱导因子(AIF)对细胞凋亡的调控[J]. 生命的化学, 2005, 25(6): 454-456. [11]Susin SK, Lorenzo HK, Zamzaml N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor[J]. Nature, 1999, 397 (6718): 441-446. DOI: 10.1038/17135 [12]Wang XC, Yang CL, Chai JJ, et al. Mechanisms of AIF-mediated apoptotic DNA degradation in caenorhabditis elegans[J]. Science, 2002, 298(5598): 1587-1592. DOI: 10.1126/science.1076194 [13]Cai JY, Yang J, Jones DP. Mitochondrial control of apoptosis: the role of cytochrome c[J]. Biochimica et Biophysica Acta, 1998, 1366: 139-149. DOI: 10.1016/S0005-2728(98)00109-1 [14]Cande C, Cecconi F, Dessen P, et al. Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death?[J]. J Cell Sci, 2002, 115(24): 4727-4734. DOI: 10.1242/jcs.00210 [15]Wang C, Wesener SR, Zhang HL, et al. An FAD-dependent pyridine nucleotide-disulfide oxidoreductase is involved in disulfide bond formation in FK228 anticancer depsipeptide[J]. Chem Biol, 2009, 16(6): 585-593. DOI: 10.1016/j.chembiol.2009.05.005