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1 微生物学通报 Microbiology China Oct. 20, 2018, 45(10): DOI: /j.microbiol.china 简报西藏株小反刍兽疫病毒 H 蛋白的原核表达及其多克隆抗体的制备于瑞嵩 1,2 高位相 1 李凤平 1,3 董世娟 1,2 李震 ( ) ( ) ( ) 1,2* 摘要 : 背景小反刍兽疫是由小反刍兽疫病毒 (Peste des petits ruminants virus,pprv) 引起的一种急性烈性接触性传染病, 严重威胁我国养羊业的发展目的原核表达 PPRV H 蛋白, 并制备其多克隆抗体方法根据 GenBank 中 PPRV 西藏株 h 基因序列, 对其进行密码子大肠杆菌偏爱性优化, 采用两步 PCR 法全化学合成全长 h 基因将测序验证正确的 h 基因克隆至原核表达载体 pet-28a pet-30a pet-32a, 转化 E. coli BL21(DE3) 并利用 IPTG 诱导 H 蛋白表达以经 SDS-PAGE 割胶纯化的重组 H 蛋白免疫新西兰大白兔制备抗 PPRV H 蛋白多克隆抗体结果重组 E. coli [pet-28a(-30a,-32a)-h] 表达的重组 H 蛋白相对分子质量分别约为和 86 kd; 诱导 7 h 时 PRRV H 蛋白表达量最高, 而且主要以包涵体形式表达 ; 重组 E. coli (pet-30a-h) 表达的 H 蛋白经 SDS-PAGE 割胶纯化后免疫新西兰大白兔制备的多抗血清能与表达的重组 H 蛋白发生特异性反应 ;ELISA 法检测抗体效价在 1: : 之间结论原核表达了 PPRV H 蛋白, 并制备了高效价的抗 H 蛋白多克隆抗体, 为进一步研究 PPRV H 蛋白的功能及 H 蛋白的线性 B 细胞表位作图奠定了基础关键词 : 小反刍兽疫病毒,H 蛋白, 原核表达, 多克隆抗体 Foundation items: National Key Research and Development Program of China (2016YFD ); National Natural Science Foundation of China ( , ); Shanghai Key Project on Agricultural Development ( ) *Corresponding author: Tel: ; zhenli60@163.com Received: December 12, 2017; Accepted: May 04, 2018; Published online ( June 13, 2018 基金项目 : (2016YFD ) ( ) ( [2015] ) * 通信作者 :Tel zhenli60@163.com 收稿日期 : 接受日期 : 网络首发日期 (

2 2184 微生物学通报 Microbiol. China Prokaryotic expression and polyclonal antibody preparation of peste des petits ruminants virus H protein YU Rui-Song 1,2 GAO Wei-Xiang 1 LI Feng-Ping 1,3 DONG Shi-Juan 1,2 LI Zhen 1,2* (1. Institute of Animal Husbandry & Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai , China) (2. Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai , China) (3. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai , China) Abstract: [Background] Peste des petits ruminants (PPR) is an acute, severe and contagious infectious disease caused by peste des petits ruminants virus (PPRV), which is endangering the sheep/goat-farming in Asian and African countries. [Objective] The objective of this study is to express PPRV H protein prokaryotically and to prepare its polyclonal antibodies. [Methods] According to the sequence of PPRV Tibet strain deposited in GenBank, h gene was optimized based on the codon usage bias of E. coli and synthesized by using two-step PCR. Then, the sequencing confirmed h gene was inserted into prokaryotic expression vectors pet-28a, pet-30a and pet-32a, respectively. The constructed recombinant plasmids pet-28a(-30a and -32a)-H were then transformed into E. coli BL21(DE3) for expression under the induction of IPTG. The expressed H protein from E. coli BL21(pET-30a-H) were purified by cutting out the aimed band from the SDS-PAGE gel. Polyclonal antibodies were prepared by immunizing New Zealand white rabbits with the purified H protein. [Results] The recombinant H protein expressed by E. coli (pet-28a (-30a, -32a)-H), with a relative molecular mass of about 70, 68 and 86 kd, respectively, mainly existed in a form of inclusion body, of which the expression level reached the maximum at 7 h after induction. The polyclonal antibodies prepared by immunizing the rabbits with the purified H protein from E. coli BL21(pET-30a-H) showed specific reactivity with expressed recombinant H protein. The titer values of the serum ranged between 1:6 400 to 1: [Conclusion] PPRV H protein was successfully expressed in prokaryotic cells, and high titer anti-h protein polyclonal antibodies were obtained, which laid a foundation for further research on the function of H protein, and the fine linear B cell epitope mapping of PPRV H protein. Keywords: Peste des petits ruminants virus, H protein, Prokaryotic expression, Ployclonal antibodies (Peste des petits ruminant PPR) (Peste des petits ruminant virus PPRV) [1] PPR PPRV PPR 100% 100% [2] PPR [3] 2007 PPR PPR 2015 PPR PPRV RNA 16 kb 3 5 -n-p/c/v-m-f-h-l 6 6 (N P M F H L) 2 (C V ) [4] PPRV H [4] PPRV H

3 : H 2185 [5] E. coli PPRV H H PPRV H B 1 材料与方法 1.1 材料菌株和质粒 (Escherichia coli E. coli) TOP 10 BL21(DE3) ( ) pmd18-t pjet1.2/blunt ( ) pet-28a (-30a -32a) 主要试剂和仪器及培养基 DNA DNA DNA Axygen ( ) ( ) PCR E. coli LB 1.2 方法 PPRV h 基因的全化学合成 GenBank PPRV H (AEH25644) DNAWorks ( nih.gov/dnaworks) E. coli PCR 55 C BamH I Hind III 64 DNA ( ) PCR h pjet1.2/blunt pjet-h PPRV H 蛋白原核表达载体的构建 BamH I Hind III PPRV h pjet-h pet-32a pet-32a-h pet-28a-h pet-30a-h PPRV h 5 Nde I Xho I (FF: 5 -CATATGATGAGCGCGCAACGGGAACGTATA AACGCCT-3 FR: 5 -CTCGAGCACCGGGTTAC AGGTCACTTCAATGC-3 ) pjet-h PCR h PCR 10 ExTaq buffer 5 μl dntp mixture 4 μl DNA 1 μl FF FR 1 μl ExTaq 1 μl ddh 2 O 50 μl PCR 98 C 2 min 98 C 15 s 55 C 30s 72 C 2 min C 5 min PCR pmd18-t Nde I Xho I h pmd18-h pet-28a pet-30a pet-28a-h pet-30a-h PPRV H 蛋白的原核表达 pet-28a-h pet-30a-h pet-30a-h E. coli BL21 LB ( 100 μg/ml 50 μg/ml) 37 C 220 r/min 1:100 9 LB 37 C 220 r/min OD IPTG 0.5 mmol/l 37 C PPRV H h 9 h 2 ml 4 C r/min 1 min SDS-PAGE PPRV H 蛋白的纯化和 Western blot 检测 E. coli BL21(pET-30a-H) 37 C 7 h 4 C r/min 1 min PBS (10 ml/g ) 20 min 4 C r/min 30 min PBS 2

4 2186 微生物学通报 Microbiol. China SDS-PAGE 0.25 mol/l KCl 10 min 2 mm 60 V 120 min 15 min PPRV H Western blot SDS-PAGE 12 V 30 min PVDF 10% 5 h ( His PPRV H 1:4 000) 4 C TBST 3 (IgG-HRP 1:20 000) 1 h TBST 4 ELC 抗 PPRV H 蛋白血清的制备 PPRV H 0.5 mg / 3 PBS 1 14 d ELISA ELISA 法检测血清的效价 (1) PPRV H (1 μg/ml) 4 C PBST 3 5% 37 C 1 h PBST 3 1:400 ( ) 37 C 1 h PBST 3 1:500 IgG 37 C 45 min PBST 3 TMB 15 min 1 mol/l OD 450 (2) 63 d 2 结果与分析 2.1 PPRV h 基因的密码子优化全化学合成及原核表达载体的构建 PPRV h h 10% PPRV h 72.6% 100% ( 1) PCR h pet-28a pet-30a pet-32a PPRV h 2.2 PPRV H 蛋白的原核表达及 Western blot 检测 SDS-PAGE pet-28a-h pet-30a-h pet-32a-h E. coli BL kd 3 E. coli PPRV H ( 2A C) 1 7 h PPRV H ( 2A C) PPRV H SDS-PAGE 2D(a) 2E(a) PPRV H H His PPRV Western blot PPRV H 3 E. coli BL21 H His PPRV [ 2D(b) 2E(b)] H ( 2E) 3 E. coli H E. coli (pet-28a-h) E. coli (pet-30a-h) H E. coli (pet-32a-h) [ 2D(a) 2E(a)] pet-30a-h E. coli BL21 7 h PPRV H PPRV H

于瑞嵩等: 西藏株小反刍兽疫病毒 H 蛋白的原核表达及其多克隆抗体的制备图1 2187 PPRV h 基因密码优化前后的序列比对 Figure 1 Sequence alignment of the wild type PPRV h gene with its codon optimized counterpart 注差异核苷酸位点以灰色阴影标出优化后 h 基因中相同的位点以.

5 于瑞嵩等: 西藏株小反刍兽疫病毒 H 蛋白的原核表达及其多克隆抗体的制备图 PPRV h 基因密码优化前后的序列比对 Figure 1 Sequence alignment of the wild type PPRV h gene with its codon optimized counterpart 注差异核苷酸位点以灰色阴影标出优化后 h 基因中相同的位点以. 代替每个氨基酸的单字母缩写位于其密码子下方. Note: Differential nucleotides were shaded with grey; The identical nucleotide in the sequence of the optimized h gene was substituted with a. ; Below the triplet code of each amino acid marked its corresponding name with one letter abbreviation.

blot (D, E) A B C pet-28a-h pet-30a-h pet-32a-h E. coli 1 E. coli 2 8 3 9 h 9. D E His PPRV Western blot PPRV H (a: SDS-PAGE b: Western blot ) 1 3 5 E.

Note: A, B, C: Recombinant E. coli transformed with pet-28a-h, pet-30a-h, pet-32a-h, respectively; 1: Precipitate of cultured recombinant E.

induced 3 9 h, respectively; 9: Precipitate of un-induced recombinant E. coli.

analysis; b: Western blot analysis); 1, 3, 5: Centrifugal supernatant of recombinant E. coli (pet-28a-h), E. coli (pet-30a-h), E.

6 2188 微生物学通报 Microbiol. China 图 2 不同诱导时间对 PPRV H 蛋白表达的影响 (A B C) 和 Western blot 验证 (D E) Figure 2 The expression time courses of PPRV H protein (A, B, C) and confirmation with Western blot (D, E) A B C pet-28a-h pet-30a-h pet-32a-h E. coli 1 E. coli h 9. D E His PPRV Western blot PPRV H (a: SDS-PAGE b: Western blot ) E. coli (pet-28a-h) E. coli (pet-30a-h) E. coli (pet-32a-h) E. coli (pet-28a-h) E. coli (pet-30a-h E. coli (pet-32a-h). PPRV H M. Note: A, B, C: Recombinant E. coli transformed with pet-28a-h, pet-30a-h, pet-32a-h, respectively; 1: Precipitate of cultured recombinant E. coli before induction; 2 8: Precipitate of recombinant E. coli induced 3 9 h, respectively; 9: Precipitate of un-induced recombinant E. coli. D, E: Western blot analysis of recombinant PPRV H protein using anti-his monoclonal antibody and goat anti-pprv serum as primary antibody (a: SDS-PAGE analysis; b: Western blot analysis); 1, 3, 5: Centrifugal supernatant of recombinant E. coli (pet-28a-h), E. coli (pet-30a-h), E. coli (pet-32a-h), respectively, broken by ultrasonication; 2, 4, 6: Precipitate of recombinant E. coli (pet-28a-h), E. coli (pet-30a-h), E. coli (pet-32a-h), respectively, broken by ultrasonication; Arrow points to the position of PPRV H protein; M: Protein marker. 2.3 PPRV H 包涵体蛋白的割胶纯化和 Western blot 检测 PPRV H SDS-PAGE SDS-PAGE His Western blot PPRV H ( 3) 2.4 抗 PPRV H 蛋白多克隆抗体的效价及特异性测定 PPRV H ELISA PPRV H 7 d 35 d ( 4) 4 1 1: : :6 400 ( 5) Western blot E. coli (pet-28a-h) E. coli (pet-30a-h) E. coli (pet-32a-h) PPRV H 3 E. coli PPRV H ( 6)

: H 2189 图 3 SDS-PAGE (A) 和 Western blot (B) 分析纯化的重组 PPRV H 蛋白 Figure 3 SDS-PAGE (A) and Western blot (B) analysis of purified PPRV H protein M 1 4 4 SDS-PAGE PPRV H.

图 6 多克隆抗体的 Western blot 鉴定 Figure 6 Western blot confirmation of the reactivity of the anti-sera with PPRV H protein A SDS-PAGE B Western blot M 1 3 5 E. coli (pet-28a-h) E. coli (pet-30a-h) E.

Note: A: SDS-PAGE analysis; B: Western blot result; M: Protein Marker; 1, 3, 5: Centrifugal supernatant of recombinant E. coli (pet-28a-h), E. coli (pet-30a-h), E.

7 : H 2189 图 3 SDS-PAGE (A) 和 Western blot (B) 分析纯化的重组 PPRV H 蛋白 Figure 3 SDS-PAGE (A) and Western blot (B) analysis of purified PPRV H protein M SDS-PAGE PPRV H. Note: M: Protein marker; 1 4: Four times purified PPRV H protein by cutting out the aimed protein from SDS-PAGE gel. 图 6 多克隆抗体的 Western blot 鉴定 Figure 6 Western blot confirmation of the reactivity of the anti-sera with PPRV H protein A SDS-PAGE B Western blot M E. coli (pet-28a-h) E. coli (pet-30a-h) E. coli (pet-32a-h) E. coli (pet-28a-h) E. coli (pet-30a-h E. coli (pet-32a-h). Note: A: SDS-PAGE analysis; B: Western blot result; M: Protein Marker; 1, 3, 5: Centrifugal supernatant of recombinant E. coli (pet-28a-h), E. coli (pet-30a-h), E. coli (pet-32a-h), respectively, broken by ultrasonication; 2, 4, 6: Precipitate of recombinant E. coli (pet-28a-h), E. coli (pet-30a-h), E. coli (pet-32a-h), respectively, broken by ultrasonication. 3 讨论与结论图 4 不同免疫时间的血清抗体水平 Figure 4 Time course of serum antibody level 图 5 血清抗体滴度的检测 Figure 5 Determination of the titers of the anti-sera H PPRV PPRV PPRV H [6-9] H PPRV h His PPRV PPRV H SDS-PAGE PPRV H H PPRV H [10] PPRV H PPRV h 10% h

8 2190 微生物学通报 Microbiol. China 3 pet (-28a -30a -32a) PPRV H h 3 PPRV H h PPRV H [10] S SDS-PAGE SDS-PAGE H PPRV H Western blot B [8,11] PPRV H H PPRV H H PPRV H PPRV H ELISA PPRV B REFERENCES [1] Baazizi R, Mahapatra M, Clarke BD, et al. Peste des petits ruminants (PPR): a neglected tropical disease in Maghreb region of North Africa and its threat to Europe[J]. PLoS One, 2017, 12(4): e [2] Adombi CM, Waqas A, Dundon WG, et al. Peste des petits ruminants in Benin: persistence of a single virus genotype in the country for over 42 years[j]. Transbound and Emerging Diseases, 2017, 64(4): [3] Baron MD, Diop B, Njeumi F, et al. Future research to underpin successful peste des petits ruminants virus (PPRV) eradication[j]. Journal of General Virology, 2017, 98(11): [4] Ye XF, Ci C. Peste des petitis ruminants and its comprehensive prevention and control measures[j]. Zhong Guo Xu Mu Shou Yi Wen Zhai, 2015, 31(2): 89 (in Chinese),. [J]., 2015, 31(2): 89 [5] Baron MD. The molecular biology of peste des petits ruminants virus[a]//munir M. Peste des Petits Ruminants Virus[M]. Berlin, Heidelberg: Springer, 2015: [6] Tao H, Sun J, Li JB, et al. Prokaryotic expression and immunoreactivity identification of peste des petits ruminants virus haemagglutinin protein[j]. Progress in Veterinary Medicine, 2014(11): 6-9 (in Chinese),,,. H [J]., 2014(11): 6-9 [7] Gao HF, Xin AG, Gao L, et al. Expression of H gene of peste des petits ruminants virus in Baculovirus[J]. Chinese Journal of Animal Infectious Diseases, 2012, 20(1): (in Chinese),,,. H [J]., 2012, 20(1): [8] Yu RS, Zhu R, Gao WX, et al. Fine mapping and conservation analysis of linear B-cell epitopes of peste des petits ruminants virus hemagglutinin protein[j]. Veterinary Microbiology, 2017, 208: [9] Herbert R, Baron J, Batten C, et al. Recombinant adenovirus expressing the haemagglutinin of Peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR[J]. Veterinary Research, 2014, 45(1): 24 [10] Tian XL, Zhao YG, Song HH, et al. Prokaryotic expression of PPRV H protein antigen epitopes[j]. Chinese Journal of Animal Health Inspection, 2009, 26(3): (in Chinese),,,. H [J]., 2009, 26(3): [11] Yu RS, Fan XM, Xu WX, et al. Fine mapping and conservation analysis of linear B-cell epitopes of peste des petits ruminants virus nucleoprotein[j]. Veterinary Microbiology, 2015, 175(1):

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇