猪囊尾蚴凋亡蛋白酶抑制剂基因的克隆与表达
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摘要
为了探究凋亡蛋白酶抑制剂基因在猪囊尾蚴生活过程中的作用,利用RT-PCR技术克隆了猪囊尾蚴凋亡蛋白酶抑制剂基因,并将其克隆至原核表达载体p ET-30a中,转化大肠杆菌BL21(D E3),经IPTG诱导后,用SDS-PAGE进行初步分析,在53 ku处出现明显的蛋白条带,与预期结果一致。W estern-blot结果表明,重组融合蛋白能够与抗H is特异性抗体发生反应。凋亡蛋白酶抑制剂基因的成功表达为后续对其功能进行研究奠定了基础。
To explore the function of apoptosis protease inhibitor gene during life cycle of Cysticercus cellulosae, the apoptosis protease inhibitor gene was cloned from C.cellulosae by RT-PCR. The c DNA fragment was subcloned into p ET-30 a vector and expressed in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that the molecular weight of expressed fusion protein was approximately 53 ku. Western-blot showed that the recombinant protein could be recognized by anti-His antibody. The study laid the foundation for the further study of the function of apoptosis protease inhibitor gene.
To explore the function of apoptosis protease inhibitor gene during life cycle of Cysticercus cellulosae, the apoptosis protease inhibitor gene was cloned from C.cellulosae by RT-PCR. The c DNA fragment was subcloned into p ET-30 a vector and expressed in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that the molecular weight of expressed fusion protein was approximately 53 ku. Western-blot showed that the recombinant protein could be recognized by anti-His antibody. The study laid the foundation for the further study of the function of apoptosis protease inhibitor gene.
引文
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[2]李海龙,景志忠,姜悦平,等.猪囊尾蚴T24基因的克隆与表达[J].中国兽医科学,2006,36(7):547-551.LI Hai-long,JING Zhi-zhong,JIANG Yue-ping,et al.Cloning and expression of T24 gene of Cysticercus cellulosae[J].Chinese Veterinary Science,2006,36(7):547-551.(in Chinese)
[3]ITO A,WANDRA T,SUBAHAR R,et al.Recent advances in basic and applied science for the control of taeniasis/cysticercosis in Asia[J].Southeast Asian J Trop Med Public Health,2002,33(Suppl 3):79-82.
[4]EDDI C,NARI A,AMANFU W,et al.Taenia solium cysticercosis/taeniosis:potential linkage with FAO activities;FAO support possibilities[J].Acta Trop,2003,87(1):145-148.
[5]COOLEY-ANDRADE O,CHEUNG K,CHEW A N,et al.Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway[J].Apoptosis,2016,[Epub ahead of print].
[6]RODRIGUES A,GARCIA H,ORTIZ P,et al.Cysteine proteases of Trypanosoma(Megatrypanum)theileri:cathepsin L-like gene sequences as targets for phylogenetic analysis,genotyping diagnosis[J].Parasitol Int,2010,59(3):318-325.
[7]WANG Q X,ZHANG S H,LUO X N,et al.Cloning and characterization of a cathepsin L-like cysteine protease from Taenia pisiformis[J].Vet Parasitol,2013,194(1):26-34.
[8]杨锐,张少华,骆学农,等.豆状带绦虫胰岛素样生长因子受体基因的克隆分析及原核表达[J].中国兽医科学,2016,46(4):403-408.YANG Rui,ZHANG Shao-hua,LUO Xue-nong,et al.Cloning,bioinformatics analysis and prokaryotic expression of insulin-like growth factor receptor gene from Taenia pisiformis[J].Chinese Veterinary Science,2016,46(4):403-408.(in Chinese)
[9]TSAI I J,ZAROWIECKI M,HOLROYD N,et al.The genomes of four tapeworm species reveal adaptations to parasitism[J].Nature,2013,496(7443):57-63.
[10]JIA W,YAN H,GUO A,et al.Complete mitochondrial genomes of Taenia multiceps,T.hydatigena and T.pisiformis:additional molecular markers for a tapeworm genus of human and animal health significance[J].BMC Genomics,2010,11:447.
[11]骆学农,郑亚东,才学鹏.猪带绦虫/囊虫病的流行现状及防制策略[J].中国病原生物学杂志,2007,2(3):230-233.LUO Xue-nong,ZHENG Ya-dong,CAI Xue-peng.Epidemic situation and preventive strategies against taeniasis/cysticercosis[J].Journalof Pathogen Biology,2007,2(3):230-233.(in Chinese)
[12]彭传林,魏川川,吴建伟,等.家蝇抗真菌肽MAF-1原核表达条件优化及活性验证[J].中国公共卫生,2015,31(11):1420-1423.PENG Chuan-lin,WEI Chuan-chuan,WU Jian-wei,et al.Optimization of expression conditions and activity verification of Musca domestica antifungal peptide-1 in prokaryotic cells[J].Chinese Journal of Public Health,2015,31(11):1420-1423.(in Chinese)
[13]王秋霞,张美英,张改平,等.猪囊尾蚴排泄分泌抗原Ts8B1蛋白原核表达条件的优化[J].华北农学报,2009,24(3):59-63.WANG Qiu-xia,ZHANG Mei-ying,ZHANG Gai-ping,et al.Optimization of prokaryotic expression of excretorysecretory antigen Ts8B1 protein gene in Cysticercus cellulosae[J].Acta Agriculturae Boreali-Sinica,2009,24(3):59-63.(in Chinese)
[14]张丹,于浩,荣义辉.戊型肝炎病毒ORF3和标签His融合真核表达载体的构建与表达[J].实用肝脏病杂志,2016,19(2):164-167.ZHANG Dan,YU Hao,RONG Yi-hui.Hepatitis E virus ORF3 and His tag fusion eukaryotic expression vector construction and expression in vitro[J].Journal of Practical Hepatology,2016,19(2):164-167.(in Chinese)