炭疽芽孢杆菌PA-LF1融合蛋白的原核表达及其反应原性研究

doi:10.16431/j.cnki.1671-7236.2023.02.025

Abstract

Abstract: 【Objective】 The purpose of this experiment was to construct the PA-LF1 fusion gene expression vector of Bacillus anthracis (B. anthracis) attenuated C40-202 strain and to detect its prokaryotic expression and regenicity,so as to provide the basis for the preparation of anthrax subunit vaccine,the diagnosis of anthrax and the detection of vaccine immune effect.【Method】 Two pairs of primers were designed based on the sequences of B. anthracis PA and LF genes in GenBank,and the PA and LF1 gene fragments were amplified from B. anthracis weakly virulent strain C40-202 by PCR.After digestion by Xho Ⅰ restriction endonuclease,the PA-LF1 fusion gene fragment was obtained by connecting the sticky ends.SWISS-MODEL analysis software was used to predict the tertiary structures of PA,LF1 and PA-LF1 fusion proteins respectively.The fusion fragments were cloned into pET32a(＋) plasmid to construct recombinant plasmid pET32a-PA-LF1,and the recombinant plasmid pET32a-PA-LF1 was transferred into E.coli BL21(DE3) competent cells for induced expression.SDS-PAGE was used to analyze the expression of the recombinant protein,and Western blotting was used to identify the fusion protein and analyze its reactogenicity.【Result】 PA, LF1 and PA-LF1 fusion genes were successfully amplified from B. anthracis weakly virulent strain C40-202.The B. anthracis PA-LF1 fusion gene expression plasmid pET32a-PA-LF1 was successfully constructed,and the tertiary structure of the fusion protein was predicted to fold to the correct spatial conformation.The recombinant plasmid was induced,purified and detected by SDS-PAGE,and the fusion protein with molecular weight of 96 ku was obtained.Western blotting showed that the purified recombinant protein had specific binding with the sheep serum immunized with anthrax spore vaccine Ⅱ,indicating that it had good reactivity.【Conclusion】 The PA-LF1 fusion protein had good reactogenicity and could provide a theoretical basis for anthrax diagnosis and vaccine immunity testing,and also could be used as a candidate component for an anthrax subunit vaccine.

Key words: Bacillus anthracis; PA-LF1 gene; clone; prokaryotic expression

CLC Number:

S852.61^＋6

LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.

References

[1] 胡凯,宰晓东,殷瑛,等.炭疽芽孢杆菌新抗原NlpC的重组表达与免疫评价[J].生物技术通讯,2020,31(6):627-633. HU K,ZAI X D,YIN Y,et al.Recombinant expression and immunological evaluation of a novel antigen NlpC from Bacillus anthracis[J]. Letters in Biotechnology,2020,31(6):627-633.(in Chinese)
[2] 田海蓉,王代英,杨志平.炭疽的流行危害及防控[J].贵州畜牧兽医,2021,5:55-57. TIAN H R,WANG D Y,YANG Z P.Epidemic hazards and prevention and control of anthrax[J]. Guizhou Journal of Animal Husbandry & Veterinary Medicine,2021,5:55-57.(in Chinese)
[3] 冯书章,刘军,孙洋,等.炭疽流行现状、流行趋势与防控策略[A].中国畜牧兽医学会动物传染病学分会第十二次人兽共患病学术研讨会暨第六届第十四次教学专业委员会论文集[C].2012. FENG S Z,LIU J,SUN Y,et al.Anthrax epidemic status,epidemic trends and prevention and control strategies[A].Proceedings of the 12th Symposium on Zoo-Zoonotic Diseases and the 14th Teaching Committee of the 6th Teaching Professional Committee of the Chinese Society of Animal Husbandry and Veterinary Medicine[C].2012.(in Chinese)
[4] MEBRATU A T,ZNABU H Z,WELDEAREGAY Y H.A retrospective survey and assessment of farmers indigenous knowledge on anthrax in and around Tanqua-Abergelle District,Northern Ethiopia[J]. Academic Journal of Animal Diseases,2015,4:10-16.
[5] WHO.Anthrax in animal and humans[Z].4th ed.Geneva:World Health Organization,2008.
[6] DOGANAY M,DEMIRASLAN H.Human anthrax as a re-emerging disease[J]. Recent Patents on Anti-infective Drug Discovery,2015,10(1):10-29.
[7] GOEL A K.Anthrax:A disease of biowarfare and public health importance[J]. World Journal of Clinical Cases,2015,3(1):20-33.
[8] FRITZE D.Taxonomy of the genus Bacillus and related genera:The aerobic endospore-forming bacteria[J]. Phytopathology,2004,94(11):1245-1248.
[9] 张玲艳,宋丽丽,贾伟娟,等.炭疽芽孢杆菌的研究进展[J].黑龙江畜牧兽医,2020,15:43-47. ZHANG L Y,SONG L L,JIA W J,et al.Research progress of Bacillus anthracis[J]. Heilongjiang Animal Science and Veterinary Medicine,2020,15:43-47.(in Chinese)
[10] 马亚妮,焦磊.炭疽芽孢杆菌抗原的研究进展[J].微生物学免疫学进展,2022,50(3):57-63. MA Y N,JIAO L.Advances in antigenic components of Bacullus anthracis[J]. Progress in Microbiology and Immunology,2022,50(3):57-63.(in Chinese)
[11] OLUDARE O,DEQUINA N,HIROSHI Y,et al.AB toxins:A paradigm switch from deadly to desirable[J]. Toxins,2010,2(7):1612-1645.
[12] ANSHUL V,MANOJ K,VIJAI P,et al.Development of a novel chimeric PA-LF antigen of Bacillus anthracis,its immunological characterization and evaluation as a future vaccine candidate in mouse model[J]. Biologicals,2019,61:38-43.
[13] SCOBIE H M,RAINEY G J A,BRADLEY K A,et al.Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor[J]. Proceedings of the National Academy of Sciences of the United States of America,2003,100(9):5170-5174.
[14] SZAROWICZ S E,DURING R L,LI W,et al. Bacillus anthracis edema toxin impairs neutrophil actin-based motility[J]. Infection and Immunity,2009,77(6):2455-2464.
[15] NEWMAN Z L,CROWN D,LEPPLA S H,et al.Anthrax lethal toxin activates the inflammasome in sensitive rat macrophages[J]. Biochemical and Biophysical Research Communications,2010,398(4):785-789.
[16] PITT M L M,LITTLE S F,IVINS B E,et al. In vitro correlate of immunity in a rabbit model of inhalational anthrax[J]. Vaccine,2001,19(32):4768-4773.
[17] ANSHUL V,GOEL A K.Development of recombinant domains of protective antigen of Bacillus anthracis and evaluation of their immune response in mouse model for use as vaccine candidates for anthrax[J]. Journal of Bioterrorism & Biodefense,2016,7(2):1-5.
[18] ANSHUL V,NIDHI P,MANOJ K,et al.An ELISA using a recombinant chimera of protective antigen and lethal factor for serodiagnosis of cutaneous anthrax in India[J]. Biologicals,2019,57:55-60.
[19] GHOSH N,GOEL A K.Anti-protective antigen IgG enzyme-linked immunosorbent assay for diagnosis of cutaneous anthrax in India[J]. Clinical and Vaccine Immunology,2012,19(8):1238-1242.
[20] GHOSH N,TOMAR I,GOEL A K,et al.A field usable qualitative anti-protective antigen enzyme-linked immunosorbent assay for serodiagnosis of human anthrax[J]. Microbiology and Immunology,2013,57(2):145-149.
[21] GHOSH N,GUNTI D,LUKKA H,et al.Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax[J]. Indian Journal of Medical Research,2015,142(2):196-204.
[22] GHOSH N,TOMAR I,LUKKA H,et al.Serodiagnosis of human cutaneous anthrax in India using an indirect anti-lethal factor IgG enzyme-linked immunosorbent assay[J]. Clinical and Vaccine Immunology,2013,20(2):282-286.
[23] BRENNEMAN K E,DOGANAY M,AKMAL A,et al.The early humoral immune response to Bacillus anthracis toxins in patients infected with cutaneous anthrax[J]. FEMS Immunology and Medical Microbiology,2011,62(2):164-172.
[24] ABDOUS M,HASANNIA S,SALMANIAN A H,et al.A new triple chimeric protein as a high immunogenic antigen against anthrax toxins:Theoretical and experimental analyses[J]. Immunopharmacology and Immunotoxicology,2019,41(1):25-31.
[25] STERN E J,UHDE K B,SHADOMY S V,et al.Conference report on public health and clinical guidelines for anthrax[J]. Emerging Infectious Diseases,2008,14(4):e1.
[26] 封纯芳.炭疽毒素蛋白的表达纯化及体外分子改良[D].武汉:华中农业大学,2009. FENG C F.The expression,purification and directed evolution in vitro of anthrax toxin[D].Wuhan:Huazhong Agricultural University,2009.(in Chinese)
[27] 王建文,焦磊,王秉翔.炭疽致死因子的结构域及功能[J].微生物学免疫学进展,2021,49(2):90-95. WANG J W,JIAO L,WANG B X.The structures and functions of the domains of anthrax lethal factor[J]. Progress in Microbiology and Immunology,2021,49(2):90-95.(in Chinese)
[28] 刘东立,马琳,张恩民,等.炭疽芽孢杆菌PA63蛋白原核表达及抗体检测间接ELISA方法的建立[J].中国病原生物学杂志,2018,13(11):1227-1231. LIU D L,MA L,ZHANG E M,et al.Study on prokaryotic of Bacillus anthracis PA63 protein and indirect ELISA[J]. Journal of Pathogen Biology,2018,13(11):1227-1231.(in Chinese)
[29] SURYANARAYANA N,VERMA M,THAVACHELVAM K,et al.Generation of a novel chimeric PALFn antigen of Bacillus anthracis and its immunological characterization in mouse model[J]. Applied Microbiology and Biotechnology,2016,100(19):8439-8451.
[30] 刘茹,李小龙,张晓倩,等.LbCas12a蛋白的原核表达及活性检测[J].中国畜牧兽医,2022,49(5):1621-1629. LIU R,LI X L,ZHANG X Q,et al.Prokaryotic expression and activity identification of LbCas12a protein[J]. China Animal Husbandry & Veterinary Medicine,2022,49(5):1621-1629.(in Chinese)

Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[3]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[4]	CHEN Jianshi, ZHANG Ning, PU Fangxiong, MIAO Jianjia, ZENG Nan, LI Bingxue. Prediction of Bacillus anthracis Carrying Prophages and Analysis of Drug Resistance and Virulence [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2573-2584.
[5]	LIU Jialong, ZHANG Yiwen, ZHANG Yueran, SUN Qijuan, CHEN Jian, HE Lei, JIA Yanyan, DING Ke, YU Zuhua. Cloning,Prokaryotic Expression and Bioinformatics Analysis of ELAVL1 Gene in Chicken [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1807-1817.
[6]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[7]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[8]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[9]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[10]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[11]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[12]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[13]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.
[14]	ZHU Lilin, WANG Houkun, CHEN Xueyang, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 310-316.
[15]	WANG Yuchen, TIAN Guangyuan, ZHOU Yaping, GUO Ting, ZHAO Hongmei, SUN Yajie, ZHAO Fengmiao, BIAN Yuchen, YU Jialiang, HAO Yongqing. Prokaryotic Expression of BPIV3 NP Protein and Its Immunoenhancing Effect on the BPIV3 Inactivated Vaccine [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3122-3130.