伽氏疏螺旋体尚志株P66基因的原核表达与鉴定

doi:10.16431/j.cnki.1671-7236.2017.01.030

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (1): 214-220.doi: 10.16431/j.cnki.1671-7236.2017.01.030

伽氏疏螺旋体尚志株P66基因的原核表达与鉴定

于培发¹, 刘志杰¹, 牛庆丽¹, 杨吉飞¹, 王振国¹, 翟斌涛¹, 潘玉平¹, 关贵全¹, 罗建勋¹, 殷宏^1,2

1. 中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室, 甘肃省动物寄生虫病重点实验室, 兰州 730046;
2. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2016-05-23 出版日期:2017-01-20 发布日期:2017-01-19
通讯作者: 殷宏 E-mail:yinhong@caas.cn
作者简介:于培发(1989-),男,山东章丘人,硕士生,研究方向:人兽共患病及兽医公共卫生学,E-mail:ypf19890123@163.com
基金资助:
农业科技创新工程（ASTIP）；国家肉牛牦牛产业技术体系（NBCISCARS-38）

Prokaryotic Expression and Identification of P66 Gene from Borrelia garinii SZ

YU Pei-fa¹, LIU Zhi-jie¹, NIU Qing-li¹, YANG Ji-fei¹, WANG Zhen-guo¹, ZHAI Bin-tao¹, PAN Yu-ping¹, GUAN Gui-quan¹, LUO Jian-xun¹, YIN Hong^1,2

1. Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2016-05-23 Online:2017-01-20 Published:2017-01-19

摘要/Abstract

摘要：

为获得伽氏疏螺旋体尚志株（B. garinii SZ）的P66蛋白，本研究从培养的螺旋体菌液中提取总RNA，反转录合成第一链cDNA，利用PCR扩增P66基因。将目的片段连接表达载体pET-30a（+），并转化大肠杆菌表达菌BL21（DE3），经PCR、双酶切及测序验证正确后，进行IPTG诱导表达和纯化，然后将纯化的融合蛋白免疫新西兰大白兔，制备多克隆抗体。SDS-PAGE结果显示，获得约70 ku的表达产物；Western blotting分析表明，表达产物与抗His标签的小鼠单克隆抗体、螺旋体鼠源阳性血清、兔抗P66多克隆抗体均能发生反应，获得的多克隆抗体也可识别天然蛋白。本试验成功表达了B. garinii SZ株P66蛋白，并制备了多克隆抗体，为后续B. garinii SZ株P66蛋白的功能研究奠定了基础。

关键词: 伽氏疏螺旋体尚志株; P66; 原核表达; 多克隆抗体

Abstract:

In order to obtain the P66 protein from Borrelia garinii (B. garinii) SZ, the first strand cDNA was synthesized based on the total RNA extracted from B. garinii SZ, and then the targeted P66 gene was amplified by PCR. The fragment was linked into the pET-30a(+) vector, and transformed into Escherichia coli BL21(DE3). After identified by PCR, double restriction enzyme digestion, and nucleotide sequencing, the recombinant protein was expressed and purified. Polyclonal antibody was then prepared from New Zealand rabbit immunized with purified recombinant protein. The recombinant protein was about 70 ku in size confirmed by SDS-PAGE, and Western blotting analysis indicated that the recombinant P66 protein could recognize the mouse monoclonal anti-His-tag, positive sera of spirochete from mouse, and anti-P66 polyclonal antibody. Additionally, the anti-P66 polyclonal antibody could recognize native P66 protein. In this study, we successfully expressed the recombinant P66 protein and obtained the anti-P66 polyclonal antibody, which provided the foundation for further functional studies of P66 protein from B. garinii SZ.

Key words: Borrelia garinii SZ; P66; prokaryotic expression; polyclonal antibody

中图分类号:

S852.63

于培发, 刘志杰, 牛庆丽, 杨吉飞, 王振国, 翟斌涛, 潘玉平, 关贵全, 罗建勋, 殷宏. 伽氏疏螺旋体尚志株P66基因的原核表达与鉴定[J]. 《中国畜牧兽医》, 2017, 44(1): 214-220.

YU Pei-fa, LIU Zhi-jie, NIU Qing-li, YANG Ji-fei, WANG Zhen-guo, ZHAI Bin-tao, PAN Yu-ping, GUAN Gui-quan, LUO Jian-xun, YIN Hong. Prokaryotic Expression and Identification of P66 Gene from Borrelia garinii SZ[J]. , 2017, 44(1): 214-220.

参考文献

[1] Steere A C, Malawista S E, Snydman D R, et al. An epidemic of oligoarticular arthritis in children and adults in three connecticut communities[J]. Arthritis Rheum, 1977, 20(1):7-17.
[2] Burgdorfer W, Barbour A G, Hayes S F, et al. Lyme disease——A tick-borne spirochetosis[J]. Science, 1982, 216(4552):1317-1319.
[3] 艾承绪,温玉欣,张永国,等. 黑龙江省海林县林区莱姆病的流行病学调查[J]. 中国公共卫生, 1987, 6(2):82-85.
[4] Wu X B, Na R H, Wei S S, et al. Distribution of tick-borne diseases in China[J]. Parasite Vector, 2013, 6(6):792-801.
[5] Bacon R, Kugeler K, Mead P. Surveillance for Lyme disease——United States,1992-2006[J]. MMWR Surveill Summ, 2008, 57(10):1-9.
[6] Biesiada G, Czepiel J, Lesniak M R, et al. Lyme disease:Review[J]. Arch Med Sci, 2012, 8(6):978-982.
[7] Pinne M, Thein M, Denker K, et al. Elimination of channel-forming activity by insertional inactivation of the p66 gene in Borrelia burgdorferi[J]. FEMS Microbiol Lett, 2007, 266(2):241-249.
[8] Coburn J. Adhesion mechanisms of the Lyme disease spirochete, Borrelia burgdorferi[J]. Curr Drug Targets-Infect Disord, 2001, 1(2):171-179.
[9] Defoe G, Coburn J. Delineation of Borrelia burgdorferi p66 sequences required for integrin α_Ⅱbβ₃ recognition[J]. Infect Immun, 2001, 69(5):3455-3459.
[10] Coburn J, Cugini C. Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent, Borrelia burgdorferi, to integrin α_Ⅴβ₃[J]. P Natl Acad Sci, 2003, 100(12):7301-7306.
[11] Bárcena-Uribarri I, Thein M, Sacher A, et al. P66 porins are present in both Lyme disease and relapsing fever spirochetes:A comparison of the biophysical properties of P66 porins from six Borrelia species[J]. BBA-Biomembranes, 2010, 1798(6):1197-1203.
[12] Bunikis J, Luke C J, Bunikiene E, et al. A surface-exposed region of a novel outer membrane protein (P66) of Borrelia spp. is variable in size and sequence[J]. J Bacteriol, 1998, 180(7):1618-1623.
[13] Cugini C, Medrano M, Schwan T G, et al. Regulation of expression of the Borrelia burgdorferi β₃-chain integrin ligand, P66, in ticks and in culture[J]. Infect Immun, 2003, 71(2):1001-1007.
[14] Chu C, Liu W, Jiang B, et al. Novel genospecies of Borrelia burgdorferi sensu lato from rodents and ticks in Southwestern China[J]. J Clin Microbiol, 2008, 46(9):3130-3133.
[15] Masuzawa T, Takada N, Kudeken M, et al. Borrelia sinica sp. nov. a lyme disease-related Borrelia species isolated in China[J]. Int J Syst Evol Micr, 2001, 51(5):1817-1824.
[16] Margos G, Chu C, Takano A, et al. Borrelia yangtzensis sp. nov. a rodent associated species in Asia is related to B. valaisiana[J]. Int J Syst Evol Micr, 2015, 65(11):3836-3840.
[17] Hao Q, Hou X, Geng Z, et al. Distribution of Borrelia burgdorferi sensu lato in China[J]. J Clin Microbiol, 2011, 49(2):647-650.
[18] 牛庆丽,杨吉飞,关贵全,等. 黑龙江尚志地区莱姆病伯氏疏螺旋体的鉴定与遗传进化分析[J]. 中国兽医科学, 2010, 40(7):661-666.
[19] Wu Q, Liu Z, Li Y, et al. Genome sequence of Borrelia garinii strain SZ, isolated in China[J]. Genome Announc, 2014, 2(4):e00010-e00014.
[20] Wu Q, Guan G, Liu Z, et al. RNA-Seq-based analysis of changes in Borrelia burgdorferi gene expression linked to pathogenicity[J]. Parasite Vector, 2015, 8(1):1-6.
[21] Schwan T G, Piesman J, Golde W T, et al. Induction of an outer surface protein on Borrelia burgdorferi during tick feeding[J]. P Natl Acad Sci, 1995, 92(7):2909-2913.
[22] Coburn J, Chege W, Magoun L, et al. Characterization of a candidate Borrelia burgdorferi β₃-chain integrin ligand identified using a phage display library[J]. Mol Microbiol, 1999, 34(5):926-940.
[23] Ntchobo H, Rothermel H, Chege W, et al. Recognition of multiple antibody epitopes throughout Borrelia burgdorferi p66, a candidate adhesin, in patients with early or late manifestations of Lyme disease[J]. Infect Immun, 2001, 69(3):1953-1956.
[24] 蒋宝贵. 我国伯氏疏螺旋体致病性及感染机制研究[D]. 北京:中国人民解放军军事医学科学院, 2007.
[25] Bunikis J, Noppa L, Ostberg Y, et al. Surface exposure and species specificity of an immunoreactive domain of a 66-kilodalton outer membrane protein (P66) of the Borrelia spp. that cause Lyme disease[J]. Infect Immun, 1996, 64(12):5111-5116.
[26] Arnaboldi P M, Dattwyler R J. Cross-reactive epitopes in Borrelia burgdorferi p66[J]. Clin Vaccine Immunol, 2015, 22(7):840-843.

伽氏疏螺旋体尚志株P66基因的原核表达与鉴定

Prokaryotic Expression and Identification of P66 Gene from Borrelia garinii SZ

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