Preparation of monoclonal antibody and subcellular localization of a-19 giardin in Giardia lamblia trophozoites
LI Wen-lin1, LI Shu-ning1, SHEN Hai-e2, ZHANGZENG Si-qi2, TIAN Xi-feng3, WANG Yang2
1.College of Clinical Medicine,Tangshan 063000,China; 2.College of life sciences,Tangshan 063210,China; 3.College of Basic Medicine,North China University of Science and Technology,Tangshan 063210,China
Abstract:The coding region ofα-19 was obtained by PCR. Transforming the recombinant vector into E.coli Rosetta (DE3) and IPTG induced expression. Immunized mice with the designed synthetic peptide by cell fusion and preparation of specific monoclonal antibody.The monoclonal antibodies(mAb) against α-19 giardin with the advantages of high affinity and specificity were pre-pared by using hybridoma technology. Western blot was used to verify the specificity and affinity of the antibodies. Subcellular localization of α-19-giardin by immunofluorescence with the most specific monoclonal antibody.The recombinant plasmid pET28a (+)-α-19 was successfully constructed by gene sequencing.The relative molecular weight of recombinant α-19 Giardia protein was 49 kDa by SDS-PAGE. Western blot results showed that the mAb secreted by a monoclonal hybridoma cell line α-19-3-7-3-5 with the highest specificity with nativeα-19 giardin protein and used for immunofluorescence analysis. The results of confocal microscopy showed that α-19 giardin was located in the ventral flagella of the trophozoites of Giardialamblia.
李汶霖, 李淑凝, 沈海娥, 章曾思琦, 田喜凤, 王洋. 蓝氏贾第鞭毛虫α-19贾第素的单克隆抗体制备与亚细胞定位研究[J]. 中国人兽共患病学报, 2020, 36(10): 801-806.
LI Wen-lin, LI Shu-ning, SHEN Hai-e, ZHANGZENG Si-qi, TIAN Xi-feng, WANG Yang. Preparation of monoclonal antibody and subcellular localization of a-19 giardin in Giardia lamblia trophozoites. Chinese Journal of Zoonoses, 2020, 36(10): 801-806.
[1] Adam RD.Biology of Giardia lamblia[J]. Clin Microbiol Rev,2001,14(3):447-475.DOI:10.1128/CMR. 14.3.447-475.2001 [2] Gardner TB, Hill DR.Treatment of giardiasis[J]. Clin Microbiol Rev,2001,14(1):114-128.DOI:10.1128/CMR. 14.1.114-128.2001 [3] WHO. The world health report 1996[R].Geneva:WHO,1996.DOI:10.1046/j.1365-3156.1997.d01-129.x [4] 王丽,夏云婷,田向,等.2014年我国南方部分地区人群蓝氏贾第鞭毛虫感染及影响因素分析[J].环境与健康杂志,2018,35(3):238-241.DOI:10.16241/j.cnki.1001-5914.2018.03.013 [5] CampanatiL, Holloschi A, Troster H,et al.Video-microscopy observations of fast dynamic processes in the protozoon Giardia lamblia[J].Cell Motil Cytoskeleton,2002,51(4):213-224.DOI:10.1002/cm.10026 [6] Cruz A,SousaMI,AzeredoZ,et al.Isolation,excystation andaxenization of Giardia lamblia isolates:in vitro susceptibility to metronidazole and albendazole[J].J Antimicrob Chemother,2003,51(4):1017-1020.DOI:10.1093/ jac/dkg150 [7] CampanatiL,Monteiro-Leal LH.The effects of the antiprotosoal drugs metronidazole and furazolidone on trophozoites of Giardislamblia(P1 strain)[J].Parasitol Res,2002,88(2):80-85.DOI:10.1007/s004360100502 [8] 余源,李巍伟,陈阳,等.蓝氏贾第鞭毛虫细胞骨架蛋白的分子生物学研究进展[J].中国病原生物学杂志,2013,8(12):1140-1147. DOI:10.13350/j.cjpb.2013.12.018 [9] Crossley R,HolbertonDV.Characterization of proteins from the cytoskeleton of Giardia lamblia[J].J Cell Sci,1983,59:8l-103. DOI:10.1111/j.2044-8287.2011.02059.x [10] Holberton DV,Ward AP.Isolation of the cytoskeleton from Giardia.Tubulin and a low-molecular-weight protein associated with microribbon strnctures[J].J Cell Sci,1981,47:139-166.DOI:10.1083/jcb.88.3.660 [11] Kim J,GooSY,ChungHJ,et al.Interaction of beta—giardinwith the Bopl protein in Giardia lamblia[J].Parasitol Res,2006,98(2):138-144.DOI:10.1007/s00436-005-0040-8 [12] NohriaA,AlonsoRA,PeattieDA.Identification and characterization of gamma-giardin and the gamma-giardin gene from Giardia lamblia[J].Mol Biochem Parasitol,1992,56:27-37.DOI:10.1016/0166-6851(92)90151-9 [13] Jenkins MC,O’Brien CN,MurphyC,et al.Antibodies to the ventral disc protein delta-giardin prevent in vitro binding of Giardia lamblia trophozoites[J].J Parasitol,2009,95(4):895-899.DOI:10.1645/GE-1851R.1 [14] Morgan RO,FernandezMP.Pilarfernandez,distinctannexin subfamilies in plants and pretists diverged prior to animal annexins and from a common ancestor[J].J Mol Evol,1997,44(2):178-188.DOI:10.1007/PL00006134 [15] 武省,李国清.蓝氏贾第虫致病机制的研究进展[J].中国动物传染病学报,2015,23(1): 64-70.DOI:10.3969/j.issn.1674-6422.2015.01.013 [16] 李汶霖,王鸿斌,李淑凝,等.蓝氏贾第鞭毛虫末端结合蛋白1基因的克隆与原核表达[J].热带病与寄生虫学,2020,18(1):21-24.DOI:10. 3969/ j. issn. 1672-2302. 2020. 01. 005 [17] 王洋,杨志宏,王沂,等.蓝氏贾第鞭毛虫α-7.l、α-ll贾第素的原核表达及抗原活性鉴定[J].中国人兽共患病学报,2012,28(5):474-478.DOI:10.3969/j.issn.1002-2694.2012.05.017 [18] 魏超君,吴玲,魏勤,等.蓝氏贾第鞭毛虫α8-贾第素基因克隆、表达与免疫反应性的鉴定[J].中国寄生虫学与寄生虫病杂志,2015, 33(3):172-175. [19] 章乐生,孙磊,胡媛.蓝氏贾第鞭毛虫单克隆抗体的制备及鉴定[J].中国寄生虫学与寄生虫病杂志,2017,35(2):160-163. [20] 王洋,王沂,杨文思,等.蓝氏贾第鞭毛虫α-4贾第素特异性多克隆抗体的制备及免疫电镜定位研究[J].中国病原生物学杂志,2012,7(12):916-919.DOI:10.13350/j.cjpb.2012.12.012 [21] 张斯雯. 贾第虫贾第素蛋白多克隆抗体的制备及免疫荧光定位研究[D].哈尔滨:东北农业大学,2017. [22] 蔡敏,黄慧聪,潘长旺.单克隆抗体在寄生虫病免疫诊断中的应用[J].国际医学寄生虫病杂志,2008, 35(2):106-110.DOI:3760/cma.j.issn.1673-4122.2008.02.012 [23] Bauer B,EngelbrechtS,Bakker-GrunwaldT,et al.Functionalidentification of alpha 1-giardin as an annexin of Giardia lamblia[J].FEMS Microbial Lett,1999,173(1):147-153.DOI:10.1111/j.1574-6968.1999.tb13496.x [24] Enrich C,Rentero C,deMuga SV,et al.Apo and calcium-bound crystal structures of eytoskeletal protein alpha-14 giardin(annexin E1)from the intestinal protozoan parasite Giardia lamblia[J].J Mol Biol,2009,385(4):1098-1112.DOI:10.1016/j.jmb.2008.11.012 [25] 王洋,余源,王卫亮,等.蓝氏贾第鞭毛虫α-4贾第素的原核表达及纯化[J].中国人兽共患病学报,2011,27(6):465-469. [26] Wei CJ,Tian XF,Adam RD,et al.Giardia lamblia:intracel1ular localization of alpha8-giardin[J].Exp Parasitol,2010,126(4):489-496.DOI:10.1016/j.exppara.2010.05.028 [27] Weiland ME, Palm JE, Griffiths WJ, et al.Charaeterisation of alpha-1 giardin:an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity[J].Int J Parasitol,2003,33(12):1341-1351. DOI:10.1016/ s0020-7519(03)00201-7 [28] 魏超君,田喜凤,卢思奇.蓝氏贾第鞭毛虫特异性细胞骨架蛋白——贾第素研究进展[J].国际医学寄生虫病杂志,2014,41(3):163-169.DOI:10.3760/cma.j.issn.1673-4122.2014.03.011