Cloning, expression and purification of Leptospira LigB antigen in Escherichia coli
Keywords:Expression, E. coli, Leptospira, LigB, purification, spirochete
Leptospira is one of the most common zoonotic diseases in the tropics and subtropics. Humans are infected by exposure to Leptospira contained water or food sources. Leptospirosis usually breaks out after the flood and causes several consequences for people and economy. Leptospirosis disease, if not rapidly detected and treated promptly, it causes serious consequences such as acute hepatitis-kidneys, meningitis and bleeding, heart and nerve complications, and severe illness can lead to death. Therefore, quick and accurate detection of Leptospira pathogen plays a very important role in Leptospirosis disease treatment. Among antigens of Leptospira, a conserved domain of LigB antigen (Leptospiral immunoglobulin-like protein) was reported that is present in the most of pathogenic serovars of Leptospira, but not in the non-pathogenic Leptospira biflexa, thus this conserved domain was used for production of Leptospirosis detection kits as well as vaccine for Leptospirosis. In order to create a kit for Leptospirosis diagonostic, especially detect anti-Leptospira antibodies in Leptospira infected serum and plasma samples, about 1kb gene fragment encoding for conserved domain of LigB (about 36 kb in molecular weight) was used as the material for producing of LigB protein by DNA recombinant technology. In this study, we present the results for cloning, expressing a conserved domain of LigB antigen in E. coli cells and purifying protein by affinity chromatography collumn. The result indicates that recombinant LigB protein was successfully expressed in E. coli Rosetta 1 and purified by Hitrap chealating collumn. The LigB protein concentration after purification reached 60 mg/L medium with 98% purity. This purified protein will be used as the materials for creating Leptospirosis kit.
Hoàng Kim Loan, Đậu Thị Việt Liên, Vũ Thị Quế Hương, Lạc Ngọc Thêm, Phan Ngọc Thảo, Lê Nhi, Bùi Chí Tâm, Phan Công Trung, Lê Thanh Tùng, Bùi Xuân Bảng, Vũ Đình Luân, Nguyễn Viết Chánh, Lê Thị Thanh Hà và Cao Thị Bảo Vân (2013) Leptospira: 10 năm (2004- 2013) khảo sát tình hình nhiễm trên người và động vật gặm nhấm ở miền nam Việt Nam, Tạp chí Y học dự phòng, tập 23, số 10(146) Số đặc biệt.
Jeanne K. Barnett, Dean Barnett, Carole A. Bolin, Theresa A. Summers, Elizabeth A. Wagar, Norman F. Cheville, Rudy A. Hartskeerl, and David A. Haake (1999) Expression and distribution of leptospiral outer membrane components during renal infection of hamsters, Infection and Immunity, 67(2), pp. 853–861.
Mohandoss Nagalingam, Sushma Rahim Assadi Thirumalesh, Triveni Kalleshamurthy, Nakkala Niharika, Vinayagamurthy Balamuruga, Rajeswari Shome, Pinaki Prasad Sengupta, Bibek Ranjan Shome, Krishnamsetty Prabhudas, Habibur Rahman, Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis, Trop Anim Health Prod (2015) 47:1329–1335, DOI 10.1007/s11250-015-0867-7.
Neida L. Conrad, Fla´via W. Cruz McBride, Je´ssica D. Souza, Marcelle M. Silveira, Samuel Fe´lix, Karla S. Mendonc, Cleiton S. Santos, Daniel A. Athanazio, Marco A. Medeiros, Mitermayer G. Reis, Odir A. Dellagostin, Alan J. A. McBride (2017) LigB subunit vaccine confers sterile immunity against challenge in the hamster model of leptospirosis, PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005441 March 16, 2017. https://doi.org/10.1371/journal.pntd.0005441 March 16, 2017.">
Nguyễn Thị Bé Mười và Hồ Thị Việt Thu (2016) Khảo sát tỷ lệ nhiễm Leptospira trên chuột (Rattus novergicus và Rattus rattus) tại tỉnh Kiên Giang, Tạp chí Khoa học Trường Đại học Cần Thơ, Số chuyên đề Nông nghiệp (2): 95-98.
Nguyễn Thị Bé Mười, Hồ Thị Việt Thu và Nguyễn Châu Nguyệt Anh (2016) Sự lưu hành của Leptospira trên chó tại tỉnh An Giang, Tạp chí Khoa học Trường Đại học Cần Thơ, Số chuyên đề Nông nghiệp (2): 91-94
Raghavan U. M. Palaniappan,Yung-Fu Chang, Fahad Hassan, Sean P. McDonough, Margaret Pough, Stephen C. Barr, Kenneth W. Simpson, Hussni O. Mohammed, Sang Shin, Patrick McDonough, Richard L. Zuerner, Jiaxin Qu and Bruce Roe (2004) Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA, Journal of Medical Microbiology (2004), 53, 975–984.
Shekatkar S, Acharya NS, Harish BN, Parija SC. (2010) Comparison of an in-house latex agglutination test with IgM ELISA and MAT in the diagnosis of leptospirosis, Indian Journal of Medical Microbiology, 28(3), p. 238.
Smits HL, van der Hoorn MA, Goris MG, Gussenhoven GC, Yersin C, Sasaki DM, Terpstra WJ, Hartskeerl RA. (2000) Simple latex agglutination assay for rapid serodiagnosis of human leptospirosis, Journal of Clinical Microbiology, 38(3), pp. 1272–1275.
T. M. A. Senthilkumar, M. Subathra, P. Ramadass, V. Ramaswamy, Serodiagnosis of bovine leptospirosis by IgG-Enzyme-Linked Immunosorbent Assay and Latex Agglutination Test, Trop Anim Health Prod (2010) 42:217–222
Tangkanakul W, Tharmaphornpil P, Plikaytis BD, Bragg S, Poonsuksombat D, Choomkasien P, Kingnate D, Ashford DA. (2000) Risk factors associated with leptospirosis in Northeastern Thailand, 1998, American Journal of Tropical Medicine and Hygiene, 63(3–4), pp. 204–208.
Veronica Barragan, Nathan Nieto, Paul Keim, and Talima Pearson (2017) Meta-analysis to estimate the load of Leptospira excreted in urine: Beyond rats as important sources of transmission in low-income rural communities, BMC Research Notes, 10(1).
Yosef Deneke, Pallab Chaudhuri, T. Sabarinath, Neha Gogia, Jonathan Lalsiamthara, K.N. Viswas, Pallab Chaudhuri, 2014, Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.01.001. http://dx.doi.org/10.1016/j.mcp.2014.01.001.">