(Luận văn thạc sĩ) nhân dòng và biểu hiện gen mã hóa protein ompl1 của chủng leptospira spp trong escherichia coli

BỘ GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - Nguyễn Văn Huy NHÂN DỊNG VÀ BIỂU HIỆN GEN MÃ HĨA PROTEIN OmpL1 CỦA CHỦNG Leptospira spp TRONG Escherichia coli LUẬN VĂN THẠC SĨ: SINH HỌC THỰC NGHIỆM Hà Nội - 2021 Luan van BỘ GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - Nguyễn Văn Huy NHÂN DÒNG VÀ BIỂU HIỆN GEN MÃ HÓA PROTEIN OmpL1 CỦA CHỦNG Leptospira spp TRONG Escherichia coli Chuyên ngành: Sinh học thực nghiệm Mã số: 8420114 LUẬN VĂN THẠC SĨ SINH HỌC THỰC NGHIỆM NGƯỜI HƯỚNG DẪN KHOA HỌC: GS.TS Nghiêm Ngọc Minh Hà Nội - 2021 Luan van i Lời cam đoan Tôi xin cam đoan cơng trình nghiên cứu tơi nhóm nghiên cứu, số liệu, kết nghiên cứu luận văn trung thực chưa có cơng bố cơng trình khác Hà nội, ngày tháng năm 2021 Tác giả Nguyễn Văn Huy Luan van ii Lời cảm ơn Để hoàn thành luận văn này, tơi xin bày tỏ lịng biết ơn sâu sắc tới GS.TS.Nghiêm Ngọc Minh, Viện Nghiên cứu hệ gen, Viện Hàn lâm Khoa học Công nghệ Việt Nam định hướng nghiên cứu, tận tình hướng dẫn, sửa luận văn tạo điều kiện hóa chất, thiết bị, kinh phí để giúp tơi thực hồn thành luận văn Tơi xin gửi lời cảm ơn chân thành tới tập thể Phòng Hệ gen học vi sinh, Viện Viện Nghiên cứu Hệ gen đặc biệt PGS.TS Võ Thị Bích Thủy bảo, giúp đỡ tận tình cho tơi q trình thực nghiệm chia kinh nghiệm chuyên môn Tôi xin gửi lời cảm ơn tới thầy cô giáo thuộc khoa Sinh học, thầy cô giáo Học viện Khoa học Công nghệ với Lãnh đạo Viện Nghiên cứu hệ gen bảo tạo điều kiện giúp đỡ học tập hoàn thành đề tài nghiên cứu Cuối cùng, xin dành lời cảm ơn đặc biệt tới gia đình, người thân bạn bè giúp đỡ, tạo điều kiện, động viên suốt thời gian học tập nghiên cứu Tôi xin chân thành cảm ơn! Hà nội, ngày tháng năm 2021 Học viên Nguyễn Văn Huy Luan van iii Danh mục ký hiệu chữ viết tắt L interrogans Leptoxspira interrogans L interrogans Leptospira interrogans L Bataviae Leptospira interrogans serovar Bataviae L Canicola Leptospira interrogans serovar Canicola L Grippotyphosa L Icterohaemorrhagiae L Pomona E.coli LB Leptospira interrogans serovar Grippotyphosa Leptospira interrogans serovar Icterohaemorrhagiae Leptospira interrogans serovar Pomona Escherichia coli Môi trường Luria Bertani PCR Polymerase Chain Reaction (phản ứng khuếch đại gen) DNA Deoxyribonucleic acid Amp Ampicillin BSA Bovine Serum Albumins (Huyết bò) TBST Tris-buffered saline với Tween 20 CBB Comassie Brilliant Blue SDS Sodium dodecyl sulfate IPTG Cơ chất Isopropyl β-D-1-thiogalactopyranoside APS Ammonium persulphate EDTA Ethylene Diamine Tetraacetace Acid OD Optical density PBS Phosphate buffer saline TEMED N,N,N’,N’-Tetramethylethylenediamine bp Base pair kb Kilobase kDa KiloDalton Luan van iv Danh mục bảng Bảng 1.1: Một số hệ biểu phổ biến 19 Bảng 2.1: Thành phần phản ứng PCR 25 Bảng 2.2: Chu trình nhiệt phản ứng PCR 25 Bảng 2.3: Trình tự mồi khuếch đại tồn gen OmpL1 25 Bảng 2.4: Thành phần phản ứng gắn gen vào vector nhân dòng 28 Bảng 2.5: Thành phần phản ứng cắt vector 30 Bảng 2.6: Thành phẩn phản ứng nối gen vào vetor 31 Bảng 2.7: Thành phần gel Acrylamide 32 Bảng 2.8: Định lượng protein phương pháp Bradford 33 Bảng 3.1: Trình tự mồi khuếch đại đoạn gen OmpL1 45 Bảng 3.2:Kết đo độ hấp phụ BSA bước sóng 595nm 57 Luan van v Danh mục hình vẽ, đồ thị Hình 1.1: Xoắn khuẩn Leptospira interrogans Hình 1.2: Con đường lây truyền bệnh Leptospirosis Hình 1.3: Sơ đồ vector pET32a 22 Hình 3.1: DNA tổng số tách chiết chủng Leptospira 37 Hình 3.2: Kết gióng hàng trình tự chuẩn chủng Leptospira interrogans 41 Hình 3.3: Kết gióng hàng trình tự chuẩn chủng Leptospira interrogans (tiếp) 42 Hình 3.4: Kết dự đốn vùng định kháng nguyên gen OmpL1 năm chủng Leptospira 43 Hình 3.5: Kết gióng hàng trình tự amino acid năm chủng Leptospira 44 Hình 3.6: Điện di đồ sản phẩm PCR đoạn gen OmpL1 45 Hình 3.7: Điện di đồ sản phẩm cắt vector pJET1.2/OmpL1 46 Hình 3.8: Điện di đồ sản phẩm plasmid 47 Hình 3.9: Điện di đồ sản phẩm cắt mở vòng vector pET32a 48 Hình 3.10: Khuẩn lạc E.coli biến nạp vector pET32a/OmpL1 môi trường LB 49 Hình 3.11: Điện di đồ sản phẩm PCR với cặp mồi T7 49 Hình 3.12: Điện di đồ sản phẩm biểu protein OmpL1 E coli BL21 50 Hình 3.13: Điện di đò sản phẩm biểu protein OmpL1 E coli BL21 điều kiện nhiệt độ nuôi cấy cảm ứng khác 52 Hình 3.14: Điện di đồ sản phẩm biểu protein OmpL1 E coli BL21 điều kiện nồng độ cảm ứng IPTG khác 54 Luan van vi Hình 3.15: Điện di đồ sản phẩm biểu protein OmpL1 E coli BL21 điều kiện thời gian cảm ứng IPTG khác 55 Hình 3.16: Điện di đồ sản phẩm sau tinh protein OmpL1 56 Hình 3.17: Đồ thị biểu diễn đường hồi quy tuyến tính 57 Hình 3.18: Chuột nhắt trắng sử dụng thử nghiệm 59 Hình 3.19: Phản ứng Western Blot kháng nguyên tái tổ hợp OmpL1 với kháng thể thu từ huyết chuột sử dụng kháng thể thứ cấp HRP Goat Anti-Mouse IgG 60 Luan van vii MỤC LỤC Lời cam đoan i Lời cảm ơn ii Danh mục ký hiệu chữ viết tắt iii Danh mục hình vẽ, đồ thị v MỞ ĐẦU CHƯƠNG TỔNG QUAN TÀI LIỆU 1.1 GIỚI THIỆU XOẮN KHUẨN LEPTOSPIRA VÀ BỆNH LEPTPSPIROSIS 1.1.1 Xoắn khuẩn Leptospira 1.1.2 Thực trạng dịch tễ bệnh Leptospirosis 1.1.2.1 Trên giới 1.1.2.2 Tại Việt Nam 1.2 NGHIÊN CỨU CHẾ TẠO PTOTEIN TÁI TỔ HỢP PHÒNG CHỐNG BỆNH LEPTOSPIROSIS 1.2.1 Đặc điểm gen xoắn khuẩn Leptospira 1.2.2 Một số nghiên cứu protein OmpL1 tái tổ hợp 11 1.2.3 Cơ chế tạo kháng thể thể xoắn khuẩn Leptospira 14 1.3 TỔNG QUAN VỀ HỆ THỐNG BIỂU HIỆN GEN 18 1.3.1 Hệ biểu Escherichia coli 18 1.3.1.1 Các hệ biểu 18 1.3.1.1 Hệ biểu Escherichia coli 19 1.3.2 Chủng biểu E coli BL21 (DE3) 20 1.3.3 Vector biểu pET32a 21 Luan van viii CHƯƠNG NGUYÊN VẬT LIỆU VÀ PHƯƠNG PHÁP 23 2.1 NGUYÊN LIỆU, THIẾT BỊ 23 2.1.1 Nguyên liệu hóa chất 23 2.1.1.1 Nguyên liệu 23 2.1.1.2 Hóa chất 23 2.1.2 Thiết bị dụng cụ nghiên cứu 24 2.1.2.1 Thiết bị 24 2.1.2.2 Dụng cụ thí nghiệm 24 2.1.2.3 Phần mềm phân tích 24 2.2 PHƯƠNG PHÁP NGHIÊN CỨU 24 2.2.1 Thiết kế mồi đặc hiệu PCR nhân gen OmpL1 24 2.2.2 PCR trực tiếp từ khuẩn lạc (Colony – PCR) 26 2.2.3 Phương pháp điện di DNA gel agarose 26 2.2.4 Xác định trình tự acid nucleic 27 2.2.5 Gắn đoạn gen vào vector nhân dòng 27 2.2.6 Biến nạp vào tế bào E coli khả biến 28 2.2.7 Tách chiết định lượng DNA plasmid 29 2.2.8 Xử lý enzyme cắt giới hạn 30 2.2.9 Gắn gen vào vector biểu pET32a 31 2.2.10 Biểu protein OmpL1 E.coli BL21 31 2.2.11 Điện di protein gel polyacrylamide có SDS (SDS-PAGE) 32 2.2.12 Phương pháp sắc ký lực 33 2.2.13 Phương pháp định lượng protein Bradford 33 Luan van 58 3.3 ĐÁNH GIÁ KHẢ NĂNG SINH MIỄN DỊCH CỦA PROTEIN TÁI TỔ HỢP OmpL1 3.3.1 Thí nghiệm sinh đáp ứng miễn dịch chuột bạch Các nghiên cứu trước rằng, động vật nhỏ chuột nhắt trắng phịng thí nghiệm khơng thể tiêm lượng lớn dung dịch thí nghiệm gây phù phổi, suy tạng dẫn đến động vật chết trước đánh giá độc tính Trong thí nghiệm này, chúng tơi sử dụng keo phèn phụ gia làm tăng khả tạo kháng thể động vật thí nghiệm, đồng thời giảm liều protein đưa vào thể chuột Dựa kết nghiên cứu tối ưu điều kiện biểu protein tái tổ hợp OmpL1, chuột nhắt trắng sử dụng cho thử nghiệm đánh giá khả kích thích tạo đáp ứng miễn dịch Số chuột chia thành nhóm, nhóm thứ gồm tiêm protein OmpL1, nhóm thứ tiêm chế phẩm vaccine Leptospira thương mại nhóm thứ tiêm PBS dủ dụng làm đối chứng âm nhóm cá thể (Hình 3.18) Cụ thể, nhóm tiêm protein OmpL1 tiêm liều 500 µl đó có chứa 0,8 mg protein OmpL1 Các bước thực theo mô tả phần phương pháp Sau toàn ba lần tiêm, ngoại trừ chuột sử dụng để lấy máu thu huyết sau tuần tiêm, tất chuột lại sống sót sau thử nghiệm có sức khỏe ổn định, khơng có dấu hiệu bất thường Những vật thu máu để tách huyết cho trình đánh giá Western Blot Như vậy, liều lượng sử dụng thử nghiệm phù hợp với động vật thí nghiệm nữa, protein tái tổ hợp OmpL1 chứng tỏ an tồn khơng gây biến chứng cho vật suốt trình thử nghiệm Luan van 59 Hình 3.18: Chuột nhắt trắng sử dụng thử nghiệm 3.3.2 Kiểm tra khả sinh miễn dịch protein tái tổ hợp OmpL1 Western blot Protein OmpL1 tái tổ hợp kiểm tra tính sinh miễn dịch phản ứng Western Blot với kháng thể thứ kháng thể thu từ huyết chuột tiêm protein tái tổ hợp OmpL1 tinh (Hình 3.19) Luan van 60 Hình 3.19: Phản ứng Western Blot kháng nguyên tái tổ hợp OmpL1 với kháng thể thu từ huyết chuột sử dụng kháng thể thứ cấp HRP Goat Anti-Mouse IgG (M): Protein Ladder; (1-4): Huyết chuột tiệm protein OmpL1 Kết Western Blot cho thấy xuất băng với kích thước tính tốn lý thuyết Điều cho thấy protein tái tổ hợp OmpL1 tiêm vào thể động vật thí nghiệm kích thích hệ thống miễn dịch vật chủ sản xuất kháng thể đặc hiệu chống lại protein Vì vậy, chúng tơi bước đầu kết luận protein tái tổ hợp OmpL1 có khả sinh miễn dịch động vật có triển vọng sử dụng để chế tạo vắc xin tái tổ hợp phòng bệnh Leptospirosis Luan van 61 CHƯƠNG KẾT LUẬN VÀ KIẾN NGHỊ 4.1 Kết luận Tạo thành công vector biểu đoạn gen đặc hiệu định kháng nguyên pET32a/OmpL1 chủng E coli BL 21 mang vector Tối ưu quy trình biểu thành công protein tái tổ hợp OmpL1 Tinh định lượng protein tái tổ hợp OmpL1 với nồng độ thu 1,653mg/ml Protein tái tổ hợp OmpL1 tạo thành gây đáp ứng miễn dịch động vật thí nghiệm 4.2 Kiến nghị Đề kết hợp với số protein tái tổ hợp khác LipL21 để nâng cao hiệu bảo hộ thử nghiệm chế tạo vắc xin tái tổ hợp Leptospira interrogans cho động vật Luan van 62 TÀI LIỆU THAM KHẢO Levett, P., 2001, Leptospirosis Clinical Microbiological Reviews, 14 Picardeau, M., 2017, Virulence of the zoonotic agent of leptospirosis: still terra incognita?, Nature Reviews Microbiology, 15(5), p 297-307 Lee, H.S., et al , 2019, Seroprevalence of leptospirosis and Japanese encephalitis in swine in ten provinces of Vietnam, PloS one, 14(8), p e0214701 Sơn Phạm Hưng, 2005, Giáo trình vi sinh vật học thú y Adler, B., 2015, History of leptospirosis and leptospira, Leptospira and Leptospirosis, Editor^Editors, Springer, p 1-9 Alston, J and H Brown, 1937, The epidemiology of Weil's disease, SAGE Publications Socolovschi, C., et al , 2011, Strikes, flooding, rats, and leptospirosis in Marseille, France, International Journal of Infectious Diseases, 15(10), p e710-e715 Brenner, D.J., et al , 1999, Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp nov and four new Leptospira genomospecies, International Journal of Systematic and Evolutionary Microbiology, 49(2), p 839-858 Bharti, A.R., et al , 2003, Leptospirosis: a zoonotic disease of global importance, The Lancet infectious diseases, 3(12), p 757-771 10 Bộ Y tế, 2014, Bệnh xoắn khuẩn vàng da Cục Y tế dự phòng 11 Levett, P.N., 2001, Clinical Microbiology Reviews, Clin MicrobioL Rev., 14(2), p 296-326 12 Faine, S., 1959, Iron as a growth requirement for pathogenic Leptospira, Microbiology, 20(2), p 246-251 13 Louvel, H., et al , 2006, 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A., et al , 2012, Primer3￢ﾀﾔnew capabilities and interfaces, Nucleic Acids Research, 40(15), p e115-e115 38 Sanger, F., S Nicklen, and A.R Coulson, 1977, DNA sequencing with chain-terminating inhibitors, Proceedings of the national academy of sciences, 74(12), p 5463-5467 Luan van 65 39 Ju, J., et al , 1995, Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis, Analytical biochemistry, 231(1), p 131-140 40 Michelsen, B.K., 1995, Transformation of Escherichia coli increases 260-fold upon inactivation of T4 DNA ligase, Analytical biochemistry, 225(1), p 172-174 41 Singh, M., et al , 2010, Plasmid DNA transformation in Escherichia Coli: effect of heat shock temperature, duration, and cold incubation of CaCl2 treated cells, International Journal of Biotechnology and Biochemistry, 6(4), p 561-568 42 El Rabey, H., et al , 2017, Isolation, cloning and sequencing of poly 3hydroxybutyrate synthesis gens from local strain of Bacillus cereus mm7 and expressing them in E coli, J Investig Genomics, 4(1), p 00056 43 Laemmli, U., 1970, SDS-page Laemmli method, Nature, 227, p 6805 44 Seixas, F.K., et al , 2007, Evaluation of different ways of presenting LipL32 to the immune system with the aim of developing a recombinant vaccine against leptospirosis, Canadian journal of microbiology, 53(4), p 472-479 45 Mahmood, T and P.-C Yang, 2012, Western blot: technique, theory, and trouble shooting, North American journal of medical sciences, 4(9), p 429 46 Dezhbord, M., et al , 2014, Molecular identification of the ompL1 gen within Leptospira interrogans standard serovars, 8(06), p 688-693 Luan van 66 CÁC CÔNG BỐ LIÊN QUAN ĐẾN LUẬN VĂN Luan van International Journal of Zoology and Animal Biology ISSN: 2639-216X MEDWIN PUBLISHERS Committed to Create Value for Researchers Expression and Purification of the Recombinant OmpL1 Protein for Potential Vaccine Production against Leptospirosis Nguyen Tuan Hung2,3, Nguyen Van Huy1,3, Nguyen Duc Hieu1, Dang Thi Quynh1,3, Vo Thi Bich Thuy1,3* and Nghiem Ngoc Minh1,3 Institute of Genome Research, Vietnam Academy of Science and Technology, Vietnam VETVACO National Veterinary Joint Stock Company, Vietnam Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Vietnam Research Article Volume Issue Received Date: November 10, 2020 Published Date: November 16, 2020 DOI: 10.23880/izab-16000252 *Corresponding author: Vo Thi Bich Thuy, Institute of Genome Research, Vietnam Academy of Science and Technology, Vietnam, Abstract Leptospirosis is a popular zoonosis caused by pathogenic strains of the Leptospira OmpL1 is the outer membrane protein present in most pathogenic Leptospira strains and consider an important factor in the immunogenicity response We describe here a simple procedure for generating recombinant OmpL1 protein resistant to Leptospira OmpL1102-316 recombinant was cloned on Escherichia coli strain DH10b and expressed in strain BL21 The purification has performed by an affinity-linked NiSepharose column with His-tag attached to this protein The recombinant protein OmpL1 induced immunological memory in laboratory rats, suggesting a potential recombinant vaccine production for using in against Leptospirosis in animals Keywords: rLipL21; Recombinant Protein Vaccine; Leptospira interrogans; Vietnam Abbreviations: OMPs: Outer Membrane Proteins; rOmpL1: Recombinant OmpL1 Protein; LB: Luria-Bertani medium; ECM: Extracellular Matrix; WHO: World Health Organization; IPTG: Isopropyl β- d-1-thiogalactopyranoside Introduction Leptospirosis, also known as acute jaundice, spreads from animals to human by the Leptospira pathogenic strains and is one of the most popular zoonoses in the world [1] According the World Health Organization (WHO) and other data sources, each year between and 10 million people suffers from Leptospirosis, with an estimated rate of 0.11/ 100,000 in temperate climates and 10-100 / 100,000 in tropical climates such as South Asia and South East Asia [2,3] Leptospirosis spreads through the skin, mucous membranes or by direct or indirect contact with mucous membranes, urine, or infected organisms In human, the disease has clinical manifestations such as latent bacterial infection, systemic toxicity syndrome, liver, kidney damage and Weil syndrome [4] In animals, it is one of the causes of stillbirth, reduced fertility, productivity and mortality, thereby causing serious economic losses [5] Currently, the commercial veterinary Leptospiral vaccines are large amount of inactivated vaccine from whole cells of Leptospira However, these vaccines have few disadvantages; they give short-term immunity [6] and elicit protection only against serovar contained in preparation The identification of genes encoding several antigens specific to pathogenic Leptospira is the decisive step in recombinant vaccine research Surface contact proteins, acting at the bacterial-host interface, are conserved between different serovars and considered suitable cross-protection vaccine candidates [7] The outer membrane proteins (OMPs) are the most important and highly conserved agent in pathogenic Leptospira strains In these, trans-membrane outer membrane protein L1 (OmpL1) is one of the most sensitive proteins for antigen-antibody immune response [8,9] The OmpL1 trans-membrane protein including 320 Expression and Purification of the Recombinant OmpL1 Protein for Potential Vaccine Production against Leptospirosis Luan van Int J Zoo Animal Biol International Journal of Zoology and Animal Biology amino acids was first reported in 1993 [10] It was shown to appear in 15 standard strains and 163 new pathogenic isolates of several Leptospira serogroups [8] Specific immunity stimulatory abilities in the animal model are due to the OmpL1’s ability to bind to extracellular matrix (ECM) and other serum components based on laminin binding and plasma fibronectin [11] Previous researches showed that recombinant OmpL1 protein (rOmpL1) could create protective immunity in animals [8,10], these indicate the high potential of rOmpL1 in vaccine production In this work, we describe the cloning and expression of a novel multi-epitope fusion protein, containing sequences of diverse previously studied OMPs of Leptospira, followed by its immunological and immune protective characterization in the hamster model of Leptospirosis Materials and Methods Materials Total DNA of five strains of Leptospira interrogans includes Leptospira interrogans serovar Bataviae (L Bataviae), Leptospira interrogans serovar Canicola (L Canicola), Leptospira interrogans serovar Grippotyphosa (L Grippotyphosa), Leptospira interrogans serovar Icterohaemorrhagiae (L Icterohaemorrhagiae) and Leptospira interrogans serovar Pomona (L Pomona), which are currently used to produce the inactivated Leptospira vaccines in Vietnam, was extraction using GeneJET Genomic DNA Purification Kit (Thermo Scientific, USA) The pJET1.2 / blunt vector (Thermo Fisher Scientific, USA) with size 2,974bp contains ampicillin resistance gene (Amp), capable of inserting a gene segment with size from 6bp to 10kb The pJET1.2/ blunt vector was used to create pJET1.2-OmpL1 plasmid Primer Design and PCR The OmpL1 sequence information was collected on the GeneBank/NCBI Based on the information of OmpL1 gene with accession numbers AY622662, using Primer3 software to design a primers pair for the OmpL1 gene xTotal DNA samples were used as a template for PCR reaction, amplifying the entire OmpL1 gene segment of five Leptospira spp Components of the reaction PCR include 10X PCR buffer (2.5µL), 2.5mM dNTP (2.5µL), 10 pmol/µL forward or reverse primer (0.5µL), 5U/µL Taq DNA polymerase (0.15µL), 200pmol/µL template (1µL), and sterile distilled water 17.85µL PCR reaction was performed with 25 cycles: denatured at 95°C for 30s, annealed at 55°C for 30s, and extended at 72°C for OmpL1 gene segments after purification and quality control were sequenced on ABI 3500 sequencing machine The obtained OmpL1 gene sequences were compared analyzed by BioEdit, BLAST and MEGA 6.06 software and selected suitable gene segment for cloning into pJET1.2 vector Cloning OmpL1 Gene OmpL1 gene was cloned from L.Batavie genome DNA with specifically designed primers The PCR product was cloned to pJET1.2 vector using CloneJET PCR Cloning Kit (Thermo Fisher) [13] Recombinant vector containing OmpL1 gene was then transformed into E coli strain DH10b strain by heat shock method Results of the transformation into E.coli of the recombinant vector pJET1.2-LipL21 were checked by PCR using OmpL1 gene-specific primers At the same time, the colonies modeling positive PCR reactions were grown in liquid LB medium supplemented with 50µg/mL of Ampicillin overnight at 37°C, shaking 200rpm Plasmid DNA extraction checked for the presence of the OmpL1 gene by cutting by restriction enzymes EcoRV and XhoI according to the manufacturer’s instructions (Thermo Fisher, USA) OmpL1 gene sequence was determined by ABI 3500 sequencing machine using pJET1.2 Forward primer The obtained nucleotide sequence was compared with the OmpL1 gene sequence using Bio edit Expression of rOmpL1 Protein in E.coli The OmpL1 gene fragment was ligated in to pET32a by T4 DNA ligase enzyme in VDNA:Vvector ratio is 2:1 Components of 20µL reaction include: 10x Buffer: 2µL, 50% PEG: 2µL, T4 ligase: 1µL, pET32a vector: 5µL and DNA fragment: 10µL The reaction mixture was incubated overnight at 4°C in order to form pET32a-OmpL1 plasmid and transformed into E.coli BL21 The transformed E.coli BL21 was cultured in liquid LB medium containing 50µg ampicillin and grown until the optical density (OD) reached 0.6 – 0.8 at 260nm At this time, 100µl IPTG (1mM) was added into medium and incubation continued until hours later at 37°C, shaking 200rpm The cells were collected by centrifugation and re-suspended by intermittently sonicating in 30 minutes in ice The soluble and insoluble fractions were isolated by centrifugation at 9000rpm for 10min Purification of the rOmpL1 Protein The recombinant OmpL1 protein contains His tag to facilitate purification His tag is found only in the recombinant protein but not in the E coli protein, makes a specific ability to adhere to the chromatographic column contained Ni2+ Recombinant OmpL1 that have specific interactions with Ni2+ will be mounted on the column, and the non-clinging or nonspecific substances are expelled Purification process uses gel, column and AKATA prime Plus automatic chromatography system (GE Healthcare) Vo Thi Bich Thuy, et al Expression and Purification of the Recombinant OmpL1 Protein for Potential Vaccine Production against Leptospirosis Int J Zoo Animal Biol 2020, 3(6): 000252 Luan van Copyright© Vo Thi Bich Thuy, et al International Journal of Zoology and Animal Biology The soluble fraction recovered from the centrifugation step described above was applied to a column containing Nisepharose gel Equilibrating the gel column with times the volume of 0.02M phosphate buffer (5mM Imidazol included) and get ready for the next purification step Protein extracts were added to the column at a rate of 0.6mL/min Unbound proteins were cursed with supplemented 0.02M phosphate buffer with the addition of 60mM Imidazol The protein fraction attached to the column was eluted with 0.02M phosphate buffer supplemented with 500mM Imidazol Fractions were analyzed by 12.5% SDS-PAGE to detect the presence of rOmpL1 and determine by Bradford method at OD 595nm Pooled proteins were dialyzed three times in phosphate buffer saline and considered to be the final production Production of Antiserum against rOmpL1 Five to eight-week-old mice were immunized with 10µg purified rOmpL1 and 100µg aluminum (Al(OH)3) The experiments were performed once a week in three weeks and mice’s blood was collected in week fourth Serum was collected in supernatant by centrifugation of blood on 6000rpm at 4°C Western blot for the Evaluation of Antibody Response Total protein from Leptospia were fractionated by 12.5% SDS-PAGE and electro-transferred to a PVDF membrane Transferring membrane was performed at 60mA/gel, 4°C, for hours, potential 100V Then, the membrane was blocked No Start End 19 24 40 10 11 12 226 251 265 285 314 AASLSA 14 170 207 Length TQKISGGVTKADIA 166 Peptide 161 148 Based on sequencing result, five Leptospira serovar’s OmpL1 genes divided into two groups, the first group was included strains of L Bataviae, L Canicola, and L Grippotyphosa with similarity about 99%, second once included strains of L Ictero haemorrhagiae and L Pomona also achieved similarity rates up to 98% Using I-TASSER and TM-score tool to integrate finding common ground between two 3D structures of OmpL1 proteins [14] (Figure 1), amino acid sequence from 102 to 316 residues (Table & Figure 2) was selected And PCR was used to amplify the gene fragment coding for OmpL1102-316 using DNA from L Canicola, as template, with the forward primer (5’- CCTGCTGATATCAGATTAATCACCCTTGATAG -3’) containing EcoRV restriction endonuclease sites (underlined) and the reverse primer (5’TTAGAGCTCGAGTTTATAACCGAATCTGTAGA -3’) containing a XhoI (underlined) restriction site 59 121 Prediction and Selection of Vaccine Epitopes and Primer Design QLGGTITKDGLDAATHYGPVRSSNTCTVGPSDPACFQNPAKPTGEGNYIGVAPRKAIPA 106 132 Results 98 with 5% skim milk-TBST for hour (or overnight at 4°C) Next, incubated membrane with primary antibody (mice’s serum in previous step) mixed in blocking solution for 1.5 hours at room temperature then washed times by TBST in minutes The membrane was continued to incubate with second antibodies in 5% nonfat dried milk-TBST solution (ratio 1:1000) for hours, following the repetition of the TBS-T wash as described above The second antibody is goat anti-mouse IgG peroxidase conjugate (Sigma, USA) 136 220 DRTTGGAINARSTKGA ESDFG VDMTW SLNGSNNIKGGYDI 248 AGAVANLLSDGTDPVTTREHVRF 300 VGKTQSVGGATNLSPF 251 267 316 Table 1: LipL21 Predicted epitopes S VTD FGY Vo Thi Bich Thuy, et al Expression and Purification of the Recombinant OmpL1 Protein for Potential Vaccine Production against Leptospirosis Int J Zoo Animal Biol 2020, 3(6): 000252 Luan van 16 5 14 23 16 Copyright© Vo Thi Bich Thuy, et al International Journal of Zoology and Animal Biology Grippotyphosa, L Icterohaemorrhagiae and L Pomona) In the assay, only one band at 38 kDa position was shown, thus antibody that collected from serum mice could recognize antigen from all of five Leptospira serovars (Figure 4) (The red is similar sequence) Figure 1: 3D construction was combined from five Leptospira serovars Figure 3: SDS-PAGE analysis of the expression of rOmpL1 protein Lane (1) (2) non induced BL21 cells; Lane (3) – (7) pellet and supernatant fractions of lysates from induced E coli BL21 cells of five Leptospira serovars Figure 2: Potential B cell epitopes for OmpL1 gene Expression and Purification of Recombinant OmpL1 The OmpL1 gen was successfully ligated into pET32a to make pET32a-OmpL1 vector then transformed into E.coli BL21 PCRs confirmed the accuracy of recombinant plasmids extracted from five colonies after culture Furthermore, PCR product sequences were completely similar to previous calculation (data not shown) The rOmpL1 was expressed from E.coli BL21 with pET32a-OmpL1 contained under IPTG induction The recombinant proteins after purification showed a single band in the gel in SDS‐PAGE assay (Figure 3), indicating that the purified proteins could become antigens for subsequent experiments After injected with purified rOmpL1, the white mice were in good-health conditions until day twenty first A Western blot assay was done with the total protein of five pathogenic Leptospira serovars (L Canicola, L Bataviae, L Figure 4: Characterization of the expressed of OmpL1 protein using Western Blot analysis M: Marker, Lane (1 - 5): Antigen-antibody reactions between five Leptospira serovars (as antigen, including L Bataviae, L Canicola, L Grippotyphosa, L Icterohaemorrhagiae and L Pomona, respectively) and antiserum (as antibody of rOmpL1) Discussion The focus of our research is producing a recombinant protein from the OmpL1 gene of pathogenic Leptospira serovars in order to make vaccine against these strains in animals We have been trying to develop an optimal process to create the protein as our goal Although combining two or more recombinant proteins in one vaccine has been shown to be effective [12], we found that it was still necessary to Vo Thi Bich Thuy, et al Expression and Purification of the Recombinant OmpL1 Protein for Potential Vaccine Production against Leptospirosis Int J Zoo Animal Biol 2020, 3(6): 000252 Luan van Copyright© Vo Thi Bich Thuy, et al International Journal of Zoology and Animal Biology evaluate the protective ability of OmpL1 independently for a more comprehensive view about serovars in Vietnam In this study, we have selected a potential sequence from OmpL1 sequences The results of sequencing showed that there were 77 differences in sequence between two Leptospira groups in the study and the difference rate of 10% Therefore, we analyzed the amino acid sequences of both groups carefully by using the 3D construction tool I-TASSER combined with TM-Score tool The results found that the similarity from amino acid 102 residues to amino acid 316 residues We have cloned OmpL1 fragment coded for rOmpL1 into pJET1.2 vector and transformed into E.coli DH10b Normally, the DNA fragments are easily degraded so should be kept in the plasmids because they are more stable Furthermore, the generation of multiple plasmid copies through culturing E coli DH10b contained the plasmid is an effective way of gene multiplication to avoid the risk of undesirable mutations in PCR We have also expressed rOmpL1 in E.coli BL21 expression system The reasons of choosing these systems are E.coli to be bacteria too, so its protein expressed will be high similar to Leptospira and increase immunological efficiency The rOmpL1 was purified by affinity tags since they help collecting large amounts of highly target proteins [14] In present, there are some widely affinity tag such as maltosebinding protein, elastin-like polypeptide tag, glutathione-Stransferase, but we used his-tags because of population Moreover, his-tag does not affect to protein activities The fact that purified rOmpL1 made immunized mice to produce antibody against OmpL1 protein of Leptospira Of course, we need to more in vivo experiments to determine ability of the protein and removing his-tag is necessary for higher amount Overall, we believe that a description of a process for expression of rOmpL1may be useful for producing a work recombinant vaccine against Leptospira Acknowledgement This research was funded by the VETVACO Join Stock Company, Vietnam Conflict of Interest: All authors have no conflict of interest References Adler B, de la Peña Moctezuma A (2010) Leptospira and leptospirosis 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al Expression and Purification of the Recombinant OmpL1 Protein for Potential Vaccine Production against Leptospirosis Int J Zoo Animal Biol 2020, 3(6): 000252 Luan van Copyright© Vo Thi Bich Thuy, et al