细粒棘球绦虫Hsp70基因的克隆、表达及抗体制备

doi:10.3864/j.issn.0578-1752.2012.12.019

摘要/Abstract

摘要： 【目的】克隆细粒棘球绦虫( Echinococcus granulosus , E.g)热休克蛋白家族基因Hsp70，在原核细胞中表达、纯化Hsp70蛋白并制备其特异性抗体。【方法】从包囊中分离原头蚴，提取RNA，RT-PCR扩增EgHsp70基因的cDNA，将其构建至原核表达载体pMAL-p2x，转化大肠杆菌BL-21菌株。通过改变诱导温度、IPTG浓度和诱导时间筛选出EgHsp70融合蛋白可溶性表达的最佳条件。并用麦芽糖亲和层析法纯化该蛋白。纯化蛋白经皮下多点注射免疫新西兰白兔制备抗血清，ELISA和Western blotting检测血清效价和特异性。【结果】 RT-PCR扩增获得EgHsp70基因的cDNA，酶切和测序结果表明EgHsp70基因已克隆入pMAL-p2x。表达条件优化实验结果筛选出Hsp70融合蛋白可溶性表达的最佳条件为：当A600为0.6时，以0.3 mmol•L-1 IPTG于30℃条件下诱导表达4 h。SDS-PAGE显示Hsp70融合蛋白相对分子质量约为68.6kD，经麦芽糖亲和层析法纯化获得了Hsp70融合蛋白，其表达量为2.5 mg•L-1。Western blotting 检测证明制备的抗血清与EgHsp70融合蛋白、原头蚴粗提抗原、E.granulosis虫体蛋白和E.granulosis虫体表面蛋白均能特异性结合，ELISA检测表明获得的抗血清效价达到 1﹕256 000。【结论】在大肠杆菌中表达并纯化获得了EgHsp70融合蛋白，并制备了高效价的兔抗EgHsp70蛋白抗血清，为研制包虫病疫苗及相关诊断试剂奠定了基础。

关键词: 细粒棘球绦虫, 热休克蛋白家族基因Hsp70, 抗血清

Abstract: 【Objective】 The objective of the experiment is to express and purify E. granulosus (Eg) heat shock protein 70 (EgHsp70) in E. coli and prepare the antibody against E. granulosus. 【Methods】 EgHsp70 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x, and the recombinant plasmid was transformed into E. coil BL-21. The soluble expression conditions of fusion protein were optimized by induction with different concentrations, of IPTG different temperatures and cultivation times. The expressed fusion protein was purified by Mal-tag Magnetic Beads. To prepare the anti-serum, New Zealand white rabbits were immunized with purified EgHsp70 protein via hypodermic and volar. Western blot was used to determine the serum’s specificity against EgHsp70 and native proteins. The serum titers were analyzed by ELISA. 【Results】 Full-length of EgHsp70 gene had an open reading frame of 765 bps encoding a protein mass of 68.6 kD. Restriction endonuclease analysis and DNA sequencing showed that EgHsp70 was cloned into the plasmid pMAL-p2x. Based on the optimization experiments, it was concluded that the best soluble expression conditions for the EgHsp70 protein are using 0.3 mmol ?L-1 IPTG when bacterial cells growing to OD600 0.6 and induced for 4 h at 30℃. ELISA and Western blotting showed that the titers of the anti-serum were above 1﹕256 000, and the anti-serum could specifically bind with EgHsp70 protein and native proteins. 【Conclusion】 The EgHsp70 fusion protein was obtained by expressing in E.coli and purifying, and the antibody against EgHsp70 was prepared with the fusion protein immunized New Zealand white rabbits. This work will provide an antigen and detection antibody for further study on the EgHsp70 function. The protein is immunogenic and can be a vaccine candidate against Echinococcus infection.

Key words: Echinococcus granulosus (Eg), heat shock protein 70(Hsp70), antiserum

赵莉, 陈皓斐, 张文宝, 马正海, 张壮志, 张旭, 古努尔?吐尔逊, 米晓云, 金映红, 薛晶, 石保新. 细粒棘球绦虫Hsp70基因的克隆、表达及抗体制备[J]. 中国农业科学, 2012, 45(12): 2491-2501.

ZHAO Li, CHEN Hao-Fei, ZHANG Wen-Bao, MA Zheng-Hai, ZHANG Zhuang-Zhi, ZHANG Xu, GU Nu-尔?Tu-尔Xun, MI Xiao-Yun, JIN Ying-Hong, XUE Jing, SHI Bao-Xin. Cloning, Prokaryotic Expression of Echinococcus granulosus Heat Shock Protein 70 and Preparation of It’s Antiserum[J]. Scientia Agricultura Sinica, 2012, 45(12): 2491-2501.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.12.019

https://www.chinaagrisci.com/CN/Y2012/V45/I12/2491

参考文献

[1]Craig P S, McManus D P, Lightowlers M W, Chabalgoity J A, Garcia H H, Gavidia C M, Gilman R H, Gonzalez A E, Lorca M, Naquira C, Nieto A, Schantz P M. Prevention and control of cystic Echinococcosis. Lancet Infection Disease, 2007, 7: 385-394.

[2]Lightowlers M W, Jensen O, Fernandez E, Iriarte J A, Woollard D J, Gauci C G, Jenkins D J, Heath D D. Vaccination trials in Australia and Argentina confirm the effectiveness of the EG95 hydatid vaccine in sheep. International Journal for Parasitology, 1999, 29:531-534.

[3]Flaherty K M, DeLuca-Flaherty C, McKay D B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature, 1990, 346(16):623-628.

[4]Revington M, Zhang Y B, Yip G N B, Kurochkin A V, Zuiderweg E R P. NMR investigations of allosteric processes in a two-domain Thermus thermophilus Hsp70 molecular chaperonee. Journal Molecular Biology, 2005, 349:163-183.

[5]Scheufler C, Brinke A r, Bourenkov G, Pegoraro S, Moroder L, Bartunik H, Hartl F U, Moarefi I. Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperonee machine. Cell, 2000, 101:199-210.

[6]Morana K A. New tricks for an old dog: the evolving world of Hsp70. New York Academy of Sciences, 2007, 1113:1-14.

[7]Tavaria M, Gabriele T, Kola I, Anderson R L. A hitchhiker’s guide to the human Hsp70 family. Cell Stress & Chaperones, 1996, 1(1): 23-28.

[8]Dang W, Hu Y H, Zhang M, Sun L. Identi?cation and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus. Fish & Shell?sh Immunology, 2010, 29:600-607.

[9]Brown W C, Norimine J, Goff W L, Suarez C E, Mcelwain T F. Prospects for recombinant vaccines against Babesia bovis and related parasites. Parasite Immunology, 2006, 28: 315-327.

[10]Brown W C. molecular approaches to elucidating innate and acquired immune responses to Babesia bovis, a protozoan parasite that causes persistent infection. Veterinary Parasitology, 2001, 101: 233-248.

[11]Brown W C, Palmer G H. Designing blood-stage vaccines against Babesia bovis and B. bigemina. Tick-borne Diseases, 1999, 15(7): 275-281.

[12]Acharya P, Kumar R, Tatu U. Chaperoning a cellular upheaval in malaria: heat shock proteins in Plasmodium falciparum. Molecular & Biochemical Parasitology, 2007, 153: 85-94.

[13]Jensen A T R, Curtis J, Montgomery J, Handman E, Theander T G. Molecular and immunological characterisation of the glucose regulated protein 78 of Leishmania Donovani. Biochimica et Biophysica Acta, 2001, 1549: 73-87.

[14]Jensen A T R, Ismail A, Gaafar A, Hassan A M E, Heander T G. Humoral and cellular immune responses to glucose regulated protein 78 a novel Leishmania donovani antigen. Tropical Medicine and International Health, 2002, 7(5):471-476.

[15]Kamiya S, Takabatake N, Fujii K, Okamura M, Iseki H, Yokoyama N, Igarashi I. Protective immunity of blood plasma fraction containing Babesia rodhaini exoantigens in mice. Journal Protozool Research, 2005, 15: 29-40.

[16]Robert J. Evolution of heat shock protein and immunity. Developmental and Comparative Immunology, 2003, 27: 449-464.

[17]Suzue K, Young R A. A djuvant-free Hsp70 fusion protein system elicits humoral and cellular immune responses to HIV-1 p24. The Journal Immunology, 1996, 156: 873-879.

[18]Zugel U, Kaufmann S H E. Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clinical Microbiology Reviews, 1999,12(1): 19-39.

[19]Udomsangpetch R, Pipitaporn B, Silamut K, Pinches R, Kyes S, Looareesuwan S, Newbold C，White N J. Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes. Medical Sciences, 2002, 99(18): 11825-11829.

[20]Shonhai A, Boshoff A, Blatch G L. The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum. Protein Science, 2007(16): 1803-1818.

[21]李三强, 崔旭红, 马灵筠, 席守民. 热休克蛋白70研究的现状及展望. 中华实用中西医杂志, 2009(22):246-247.

Li S Q, Cui X H, Ma L J, Xi S M. A review and prospect on heat shock protein 70. Chinese Journal of the Practical Chinese with Modem Medicine, 2009, 22(4):246-247.(in Chinese)

[22]Sreedhar A S, Vanathi P, Aithankar K R. Stress proteins in biology and medicine: evolution, adaptation and clinical evaluation. International Journal of Pharmacology and Biological Sciences, 2010, 1(3): 1-36.

[23]谢挺. 热休克蛋白70的研究. 中华国际医学杂志, 2003，3(5):441-444.

Xie T. The resease of heat shock protein 70. Chinese International Journal Medical, 2003, 3(5): 441-444. (in Chinese)

[24]丁淑琴, 赵嘉庆, 王娅娜, 王健, 赵巍. 细粒棘球蚴热休克蛋白70基因的克隆和序列分析, 宁夏医学杂志, 2005, 27(8): 507-509.

Ding S Q, Zhao J Q, Wang Y N, Wang J, Zhao W. Cloning and sequence analyzing of the Hsp70 gene from Echinococcus granulosus. Ningxia Medical Journal, 2005, 27(8): 507-509. (in Chinese)

[25]Kiang J G， Tsokos G C. Heat shock protein 70 kDa: molecular biology, biochemistry and physiology. Pharmacology & Therapeutics, 1998(80):184-185.

[26]Koziol U, Iriarte A, Castillo E, Soto J, Bello G, Cajarville A, Roche L, Marín M. Characterization of a putative Hsp70 pseudogene transcribed in protoscoleces and adult worms of Echinococcus granulosus. Gene, 2009, 443:1-11.

[27]Brocchieri L, de Macario E C， Macario A J L. Hsp70 genes in the human genome: conservation and differentiation patterns predict a wide array of overlapping and specialized functions. BMC Evolutionary Biology, 2008, 8:19.

[28]Kumar N, Nagasawa H, Sacci Jr J B, Sina B J, Aikawa M, Atkinson C, Uparanukraw P, Kubiak L B, Azad A F, Hollingdale M R. Expression of members of the heat-shock protein 70 family in the exoerythrocytic stages of Plasmodium berghei and Plasmodium falciparum. Parasitology Research, 1993, 79: 109-113.

[29]丁淑琴, 王洁, 王淑静, 张焱, 赵巍. 细粒棘球蚴2Hsp70重组质粒的构建、原核表达、纯化和初步鉴定. 中国人兽共患病学报, 2006, 22(8): 764-766.

Ding S Q, Wang J, Wang S J, Zhang Y, Zhao W. Echinococcus granulosus 2 heat shock protein 70 gene: construction of tecombinant plasmid, prokaryotic expression, purification and characterization. Chinese Journal of Zoonoses, 2006, 22(8): 764-766.(in Chinese)

[30]丁淑琴, 张爱君, 赵巍. 细粒棘球蚴热休克蛋白70重组抗原的免疫学特性研究，宁夏医学院学报，2007, 29(5):454-459.

Ding S Q, Zhang A J, Zhao W. Immunogenitity identifition of Echinococcus Granulosus recombinant heat shock protein 70. Journal of Ningxia Medical College, 2007, 29(5):454-459. (in Chinese)

[31]Nemoto T K, Fukuma Y, Itoh H, Takagi T, Ono T. A disul?de bridge mediated by cysteine 574 is formed in the dimer of the 70-kDa heat shock protein. The Japanese Biochemical Society, 2006, 139(4): 677-687.