Abstract：To analyze the structural characteristics of the Rap2B gene from Clonorchis sinensis by bioinformatics and to clone and express this gene through genetic engineering methods in order to obtain the corresponding recombinant protein.The structural and functional characteristics of the Rap2B protein were analyzed by using the related bioinformatic softwares.Using the full-length cDNA plasmid clone(Noc009e03) as template,the coding region of Rap2B gene sequence was amplified with PCR and cloned into the prokaryotic expression vector pET-28a(+) and then expressed in E.coli BL21 through IPIG induction.The recombinant protein expressed was purified by Ni-IDA affinity chromatography and detected by SDS-PAGE and Western blotting.It was demonstrated that the size of this gene was 847 bp in length,encoding 141 amino acids and its theoretical molecular weight was 15851.1 daltons.As demonstrated by PCR,double enzyme digestion and DNA sequencing,the recombinant expression plasmid pET-28a(+)-Rap2B was successively constructed and the Rap2B-like gene was highly expressed in E.coli BL21.The recombinant protein was obtained through purification with His affinity chromatography and could react specifically with the serum of rats infected with C.sinensis.Through these investigations,the Rap2B protein may be predicted as a member of the Ras oncogene family protein and is potential in the carcinogenic process of C.sinensis.
[1]Lun ZR,Gasser RB,Lai DH,et al.Clonorchiasis:a keyfood-borne zoonosis in China〔J〕.Lancet Infect Dis,2005,5(1):31-41.
[2]Stunell H,Buckley O,Geoghegan T,et al.Recurrent pyogeniccholangitis due to chronicinfestation withClonorchissinensis〔J〕.Eur Radiol,2006,16(11):2612-2614.
[3]吴德,余新炳,吴忠道.华支睾吸虫病的流行概况〔J〕.热带医学杂志,2002,2(3):27-279.
[4]Srivatanakul P,Sriplung H,Deerasamee S.Epidemiology of liv-er cancer:an over view〔J〕.Asian Pac J Cancer Prev,2004,5(2):118-225.
[5]Ri m HJ.Clonorchiasis:an update〔J〕.J Hel minthol,2005,79(3):269-81.
[6]An Q,Pacyna-Gengelbach M,Schluns K,et al.Identification ofdifferentially expressedgenes in i mmortalized human bronchialepithelial cell line as a model forinvitrostudy oflungcarcinogene-sis〔J〕.Int J Cancer,2003,103(2):194-204.
[7]Mascaux C,Iannino N,Martin B,et al.The role of RAS onco-gene in survival of patientswith lung cancer:a systematic reviewof theliterature with meta-analysis〔J〕.British Journal of Cancer,2005,92(1):131-139.
[8]赵玉利,胡旭初,徐劲,等.华支睾吸虫CsSCCRO基因的克隆表达和蛋白纯化〔J〕.中国人兽共患病学报,2006,22(10):906-908.
[9]胡旭初,赵宇,黄灿,等.华支睾吸虫多药物/代谢产物外排蛋白样基因的识别和生物信学分析〔J〕.中国人兽共患病学报,2008,24(2):104-108.
[10]J.萨姆布鲁克,EF.里奇,T.曼尼阿蒂斯.分子克隆实验指南〔M〕.2版,北京:科学出版社,1992:852-858.
[11]谢红艳,胡旭初,徐劲,等.华支睾吸虫Rho GTPase样基因的识别、克隆表达与免疫鉴定〔J〕.中国人兽共患病学报,2006,22(6):561-564.
[12]李舒,郭莲怡,陈洋.幽门螺杆菌感染与胃癌和癌前病变p53、ras基因表达〔J〕.辽宁医学院学报,2008,29(5):410-412.
[13]Uechi Y,Bayarjargal M,Umikawa M,et al.Rap2 function re-quire pal mitoylation and recycling endosome localization〔J〕.Bio-chemical and Biophysical Research Communications,2009,378(4):732-737.
[14]Ohmstede CA,Farrell FX,Reep BR,et al.Rap2B:a RAS-related GTP-binding protein from platelets〔J〕.Biochemistry,1990,87(17):6527-6531.
[15]Torti M,Lapetina E G.Structure and function of Rap proteinsin human platelets〔J〕.Thromb Haemost,1994,71(5):533-543.
[16]Greco F,Ciana A,Pietra D,et al.Rap2,but not Rap1 GTPaseis expressedin human red blood cells andis involvedin vesicula-tion〔J〕.Biochi m Biophys Acta,2006,1763(3):330-335.
[17]Shih A,Davis FB,Lin HY,et al.Resveratrol induces apopto-sis inthyroid cancer cell lines via MAPKand p3-dependent mech-anism〔J〕.Clin Endocrinol Metab,2002,87(3):1223-1232.
[18]张巧,陈景涛,吴卫东,等.肺癌组织中RAP2B的表达〔J〕.癌变.畸变.突变,2007,19(6):466-468.
[19]Liu Y,Sun W,Zhang K,et al.Identification of genes differen-tially expressedin human pri mary lung squamous cell carcinoma〔J〕.Lung Cancer,2007,56(3):307-17.
