Cloning,Sequence Analysis and Prokaryotic Expression of CPAF Gene of Chlamydia abortus

doi:10.7668/hbnxb.2018.03.014

Abstract

Abstract: In order to investigate the immunogenicity of Chlamydial protease like activity factor(CPAF)of C. abortus and establish a rapid method for detection of Chlamydia abortus (C. abortus),a pair of specific primers for CPAF of C. abortus were designed and synthesized. The gene was amplified by PCR from C. abortus reference strain. The obtained gene sequence was sequenced and analyzed by bioinformatics software. The obtained gene of 1 809 bp was inserted into plasmid pET-30a. The recombinant plasmid was identified by digesting and sequencing. Then the successfully constructed recombinant plasmid was transformed into E.coli BL21(DE3)strain as host cell for expression of the recombinant protein by IPTG induction. The production was detected by SDS-PAGE and Western Blot analysis. The CPAF gene of Chlamydia abortus was successfully cloned and analyzed. This gene was likely to be located in the cytoplasm of host cell,playing a role as a proteolytic enzyme with several potential antigenic epitope. As shown by SDS-PAGE and Western Blot analysis,a 67.6 ku recombinant protein was expressed by IPTG induction,which could combine with anti-His-tag monoclonal antibody and C. abortus positive serum with well reactiongenicity. These results suggested that the recombinant CPAF could be used as a potential antigen for serological tests and vaccine development of C. abortus.

Key words: Chlamydia abortus, CPAF, Clone, Sequence analysis, Prokaryotic expression

CLC Number:

S855
Q78

LIU Ping, MA Xiaoxia, ZHOU Xiaokai, MA Peng, CHANG Qiuyan, LI Linjie, LI Linghao, MA Zhongren. Cloning,Sequence Analysis and Prokaryotic Expression of CPAF Gene of Chlamydia abortus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(3): 86-91. doi: 10.7668/hbnxb.2018.03.014.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2018.03.014

http://www.hbnxb.net/EN/Y2018/V33/I3/86

References

[1] Sachse K,Bavoil P M,Kaltenboeck B,et al. Emendation of the family Chlamydiaceae:proposal of a single genus,Chlamydia,to include all currently recognized species[J]. Systematic and Applied Microbiology,2015,38(2):99-103.
[2] Hybiske K,Stephens R S. Mechanisms of host cell exit by the intracellular bacterium Chlamydia[J]. Proceedings of the National Academy of Sciences of the United States of America,2007,104(27):11430-11435.
[3] Hybiske K,Stephens R S. Mechanisms of Chlamydia trachomatis entry into nonphagocytic cells[J]. Infection and Immunity,2007,75(8):3925-3934.
[4] Zhong G,Fan P,Ji H,et al. Identification of a Chlamydial protease-like activity factor responsible for the degradation of host transcription factors[J]. The Journal of Experimental Medicine,2001,193(8):935-942.
[5] Zhong G. Chlamydia trachomatis secretion of proteases for manipulating host signaling pathways[J]. Frontiers in Microbiology,2011,2:14.
[6] Jorgensen I,Bednar M M,Amin V,et al. The Chlamydia protease CPAF regulates host and bacterial proteins to maintain pathogen vacuole integrity and promote virulence[J]. Cell Host & Microbe,2011,10(1):21-32.
[7] Yang Z,Tang L,Zhou Z,et al. Neutralizing antichlamydial activity of complement by Chlamydia-secreted protease CPAF[J]. Microbes and Infection,2016,18(11):669-674.
[8] Tang L,Chen J,Zhou Z,et al. Chlamydia-secreted protease CPAF degrades host antimicrobial peptides[J]. Microbes and Infection,2015,17(6):402-408.
[9] 王华丽,吴移谋,郑江花,等. 肺炎嗜衣原体CPAF重组蛋白致小鼠肺组织炎症及对炎症细胞因子的影响[J]. 中国免疫学杂志,2011,27(4):308-311.
[10] Zheng J,Ding T,Chen Z,et al. Preparation and evaluation of monoclonal antibodies against chlamydial protease-like activity factor to detect Chlamydia pneumoniae antigen in early pediatric pneumonia[J]. European Journal of Clinical Microbiology & Infectious Diseases,2015,34(7):1319-1326.
[11] Barati S,Moori-Bakhtiari N,Najafabadi M G,et al. The role of zoonotic chlamydial agents in ruminants abortion[J]. Iranian Journal of Microbiology,2017,9(5):288-294.
[12] Softic A,Asmare K,Granquist E G,et al. The serostatus of Brucella spp., Chlamydia abortus,Coxiella burnetii and Neospora caninum in cattle in three cantons in Bosnia and Herzegovina[J]. BMC Veterinary Research,2018,14(1):40.
[13] Walder G,Hotzel H,Brezinka C,et al. An unusual cause of sepsis during pregnancy:recognizing infection with Chlamydophila abortus[J]. Obstetrics and Gynecology,2005,106(5 Pt 2):1215-1217.
[14] Li Z,Cao X,Fu B,et al. Identification and characterization of Chlamydia abortus isolates from yaks in Qinghai,China[J]. BioMed Research International,2015:658519.
[15] Hu S F,Li F,Zheng W B,et al. Seroprevalence and risk factors of Chlamydia abortus infection in goats in Hunan province,subtropical China[J]. Vector Borne and Zoonotic Diseases,2018:2183.
[16] Entrican G,Wattegedera S,Wheelhouse N,et al. Immunological paradigms and the pathogenesis of ovine chlamydial abortion[J]. American Journal of Reproductive Immunology,2010,64(4):287-294.
[17] Li Z,Cai J,Cao X,et al. Whole-Genome sequence of Chlamydia abortus Strain GN6 isolated from aborted yak fetus[J]. Genome Announcements,2017,35(5):e00817-e00893.
[18] Wheelhouse N,Sait M,Wilson K,et al. Expression patterns of five polymorphic membrane proteins during the Chlamydia abortus developmental cycle[J]. Veterinary Microbiology,2012,160(3/4):525-529.
[19] Chisu V,Porcu R,Tanda A,et al. First isolation and characterization of Chlamydophila abortus from abortion tissues of sheep in Sardinia,Italy[J]. Veterinaria Italiana,2013,49(4):331-334.
[20] Hireche S,Bouaziz O,Djenna D,et al. Seroprevalence and risk factors associated with Chlamydophila spp. infection in ewes in the Northeast of Algeria[J]. Tropical Animal Health and Production,2014,46(2):467-473.
[21] Pirbhai M,Dong F,Zhong Y,et al. The secreted protease factor CPAF is responsible for degrading pro-apoptotic BH3-only proteins in Chlamydia trachomatis -infected cells[J]. The Journal of Biological Chemistry,2006,281(42):31495-31501.
[22] Fan P,Dong F,Huang Y,et al. Chlamydia pneumoniae secretion of a protease-like activity factor for degrading host cell transcription factors required for[correction of factors is required for]major histocompatibility complex antigen expression[J]. Infection and Immunity,2002,70(1):345-349.
[23] Chen A L,Johnson K A,Lee J K,et al. CPAF:a chlamydial protease in search of an authentic substrate[J]. PLoS Pathogens,2012,8(8):e1002842.
[24] Forsbach-Birk V,Foddis C,Simnacher U,et al. Profiling antibody responses to infections by Chlamydia abortus enables identification of potential virulence factors and candidates for serodiagnosis[J]. PLoS One,2013,8(11):e80310.
[25] 周继章,曹小安,邱昌庆,等. 动物衣原体病诊断技术[S]. 中华人民共和国农业行业标准,2015,562/T-2015.
[26] Hireche S,Ababneh M M,Bouaziz O,et al. Seroprevalence and molecular characterization of Chlamydia abortus in frozen fetal and placental tissues of aborting ewes in northeastern Algeria[J]. Tropical Animal Health and Production,2016,48(2):255-262.

Please choose a citation manager

Content to export