CONSTRUCTION, EXPRESSION AND PURIFICATION OF BRUCELLA SPP. RECOMBINANT PROTEINS L7/L12 AND SODC IN E. COLI-Reference-Cited by-同舟云学术

CONSTRUCTION, EXPRESSION AND PURIFICATION OF BRUCELLA SPP. RECOMBINANT PROTEINS L7/L12 AND SODC IN E. COLI

Published:2020-04-15 Issue:338 Volume:2 Page:20-30
ISSN:2518-1629
Container-title:Series of biological and medical
language:
Short-container-title:

Author:

Tailakova E. T.¹^ORCID,Sadikaliyeva S. О.²^ORCID,Shynybekova G. O.³^ORCID,Abubakirova A. K.⁴^ORCID,Sultankulova K. T.⁵^ORCID,Chervyakova O. V.⁶^ORCID

Affiliation:

1. master of biology, researcher of the laboratory of “Molecular biology and genetic engeneering”, Research Institute for Biological Safety Problems, Korday district, Zhambyl region, Gvardeiskiy v., Kazakhstan; tailakova_86@mail.ru; https://orcid.org/0000-0003-3987-7451

2. master of chemistry, researcher of the laboratory of “Molecular biology and genetic engeneering”, Research Institute for Biological Safety Problems, Korday district, Zhambyl region, Gvardeiskiy v., Kazakhstan; sadikalieva86@mail.ru; https://orcid.org/0000-0003-4953-3628

3. master of biology, senior assistant of the laboratory of “Molecular biology and genetic engeneering”, Research Institute for Biological Safety Problems, Korday district, Zhambyl region, Gvardeiskiy v., Kazakhstan; gaukhar_1988@bk.ru; https://orcid.org/0000-0002-6976-1540

4. master of veterinary sciences, senior assistant of the laboratory of “Molecular biology and genetic engeneering”, Research Institute for Biological Safety Problems, Korday district, Zhambyl region, Gvardeiskiy v., Kazakhstan; aigerim_abubakirova94@mail.ru; https://orcid.org/0000-0003-3601-8977

5. candidate of biological sciences, professor, head of the laboratory of “Molecular biology and genetic engeneering”, Research Institute for Biological Safety Problems, Korday district, Zhambyl region, Gvardeiskiy v., Kazakhstan; sultankul70@mail.ru; https://orcid.org/0000-0002-1332-1247

6. candidate of biological sciences, leading researcher of the laboratory of “Molecular biology and genetic engeneering”, Research Institute for Biological Safety Problems, Korday district, Zhambyl region, Gvardeiskiy v., Kazakhstan; ovch@mail.ru; https://orcid.org/0000-0002-7954-6246

Abstract

Brucellosis is still an important public health problem as long as natural reservoirs of infection exist. Currently, live attenuated vaccines based on strains S19, RB51 and Rev1 are used for the prevention of brucellosis in animals, the main disadvantage of which is virulence for humans. However, animal immunization programs should be implemented to reduce the incidence of humans. The development of safe and effective new generation vaccines using “omix” technology is a promising direction of vaccinology. A number of immunogenic Brucella proteins that elicit both a humoral and cellular immune response has been identified. The aim of these research was to optimize the expression and purification conditions of the Brucella spp. recombinant proteins L7/L12 and SodC. As a result, expressing plasmids pET/Br-L7/L12 and pET/Br-SodC were obtained. The parameters of target genes expression in E. coli were established and the method for purification of recombinant proteins was optimized. Purification of the L7/L12 protein was performed under hybrid conditions on HisPur agarose using a binding buffer containing 6 M guanidine hydrochloride, a wash buffer with 20 mM imidazole and an elution buffer with 300 mM imidazole. Protein SodC was purified under denaturing conditions with the addition of 1 % Triton X-100 and 1 % sodium deoxycholate to the lysis buffer. Inclusions were solubilized with a buffer containing 8 M urea and 5 mM imidazole. The target protein was eluted from HisPur agarose with buffer containing 8 M urea and 100 mM imidazole. The use of modified purification protocols made it possible to obtain purified recombinant proteins with a yield of 13 mg/L for the L7/L12 protein and 10 mg/L for the protein SodC, respectively. The specificity of the proteins was confirmed by a Western blot. Immunization of mice with recombinant proteins led to the production of specific antibodies, the titer of which in ELISA was 1:20480 and 1:20480, respectively.

Publisher

National Academy of Sciences of the Republic of Kazakshtan

Link

http://biological-medical.kz/images/pdf/b20202/20-30.pdf

Reference26 articles.

1. Ivanov N.P. (2013) Diseases of ruminants, pigs and horses, diseases of birds, carnivores and fur-bearing animals, bees, fish, little-known diseases and slow infections [Bolezni zhvachnyh zhivotnyh, svinej i loshadej, bolezni ptic, plotojadnyh i pushnyh zverej, pchel, ryb, maloizvestnye bolezni i medlennye infekcii]. Infectious animal diseases: tutorial [Infektsionnye bolezni zhivotnykh: uchebnoe posobie], Almaty: Nur-print. ISBN: 978-601-241-370-0 (in Russ.).

2. Ivanov N.P. (2007) Animal brucellosis and control measures: tutorial [Brutsellez zhivotnykh i mery borby s nim: uchebnoe posobie], Almaty: Atamura (in Russ.).

3. Janyshev A.A. (2007) Cattle and small brucellosis in the Southern Federal District: epizootological monitoring and improvement of antiepizootic measures [Brucelljoz krupnogo i melkogo rogatogo skota v Juzhnom Federal'nom okruge: epizootologicheskij monitoring i sovershenstvovanie protivoepizooticheskih meroprijatij], Avtoref. dis. k.v.n., M., 21 p. (in Russ.).

4. Enright F.M., Nielsen K. and Duncan J.R. (1990) The pathogenesis and pathobiology of Brucella infection in domestic animals. Animal Brucellosis, CRC Press, Boca Raton, FL, P. 301–320. ISBN:0849358787 (in Eng.).

5. Sattarov A.I. (2005) Brucellosis in the Republic of Kazakhstan, Materials of the international scientific-practical conference dedicated to the 100th anniversary of KazNIVI [Brucellez v Respublike Kazahstan, Materialy mezhdunarodnoj nauchno-prakticheskoj konferencii, posvjashhennoj 100 – letiju KazNIVI], Almaty, P. 237-241 (in Russ.).