طراحی و ایجاد موتاسیون در ژن wbk A بروسلا آبورتوس S19 به روش Overlap Extension PCR

نویسندگان

1 گروه میکروبیولوژی، دانشکده دامپزشکی دانشگاه تهران

2 گروه فناوری زیستی، دانشگاه صنعتی مالک اشتر

چکیده

زمینه مطالعه: ایجاد موتاسیون در جایگاه اختصاصی می‌تواند یکی از روش‌های کارآمد جهت بررسی ویژگی و خواص تنظیمی ژن‌های گوناگون باشد. ﺑﺮوﺳﻠﻮز از ﻣﻬﻢ ﺗﺮﯾﻦ ﺑﯿﻤﺎریهای ﻋﻔﻮﻧﯽ ﻣﺸﺘﺮک ﺑﯿﻦ اﻧﺴﺎن و دام اﺳﺖ ﻛﻪ ﻣﻨﺠﺮ ﺑﻪ بروز ﺿﺮرهاى اﻗﺘﺼﺎدى ﻓﺮاواﻧﻰ ﻣﻰ ﺷﻮد. ﺑﻨﺎﺑﺮاﯾﻦ ﺷﻨﺎﺳﺎﯾﯽ ﻋﻮاﻣﻞ پاتوژن و اﯾﻤﻨﯽ زا در جنس ﺑﺮوﺳﻼ ﺑﻪ ﻋﻨﻮان راهگشای ﺟﻬﺖ ﮐﻨﺘﺮل اﯾﻦ ﻣﻌﻀﻞ ﺑﻬﺪاﺷﺘﯽ ﻣﻄﺮح ﻣﯽ‌ﺑﺎﺷﺪ. هدف: با توجه به اهمیت جهش هدفدار در شناسایی ساختار ژنوم و وجود روش‌های متعدد جهت دست یابی به این هدف، روش Overlap Extension PCR به عنوان یک تکنیک اصلاح شده جهت حذف و جایگزینی ژن هدف معرفی می‌شود. روش کار: جهت انجام این تحقیق، با انجام دو مرحله PCR با استفاده از پرایمرهای اختصاصی، قطعات بالادست و پایین دست ژن هدف از ژنوم باکتری و کاست مقاومت آنتی بیوتیکی از پلاسمیدa(+)رpET28، تکثیر یافته و به یکدیگر اتصال یافتند. قطعه حاصله با استفاده از آنزیم‌های محدودالاثر که توالی آنها در انتهای ´5 پرایمرهای خارجی قرار داده شده است، در جایگاه اختصاصی از پلاسمید (-) pBluescriptIISKهمسانه سازی شده و پس از حصول اطمینان از عدم ایجاد جهش خودبخودی در حین مراحل PCR، با استفاده از روش الکتروپوریشن به داخل ژنوم بروسلا آبورتوس انتقال یافت. نتایج: همسانه سازی محصول PCR اتصالی که بدون بروز تغییر در توالی نوکلئوتیدی حاصل شده بود، داخل پلاسمید (-) pBluescriptIISK انجام شد و پس از انتقال الکتریکی پلاسمید به ژنوم بروسلا آبورتوس، طی عمل ریکامبینیشن باعث جهش در ژن مورد نظر گردید. نتیجه گیری نهایی: نتایج این مطالعه نشان می‌دهد که Overlap Extension PCR یک تکنیک بهینه و اصلاح شده به منظور ایجاد جهش در ساختار ژنوم باکتری بوده و به راحتی می‌تواند در خانواده بروسلا استفاده گردد.

کلیدواژه‌ها


عنوان مقاله [English]

Construction of mutant WbkA gene in Brucella abortus S19 by overlap extension PCR

نویسندگان [English]

  • Solmaz Naserli 1
  • Taghi Zahraei salehi 1
  • Bahar Nayeri fassayi 1
  • Alireza Saeedinia 2
  • Iraj Ashrafi tamami 1
1 Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran
2 Department of Genetics, Sciences and Biotechnology Research Center, Mallek-Ashtar University of Technology, Tehran-Iran
چکیده [English]

BACKGROUND: Causing site direct mutation can be one of the efficient methods to evaluate the characteristics and properties of various genes. Brucellosis is the most common zoonotic infectious disease that would cause great economic losses. Thus, recognition of pathogenic and immunogenic factors in the genus Brucella can lead to control this health problem. Objectives: Considering the importance of site direct mutation in identification of genome structure and numerous ways to achieve this goal, Overlap Extension PCR is introduced as an improved technique for the removal and replacement of the gene target. Methods: For this study, with two-step PCR using specific primers, upstream and downstream fragments from target gene and antibiotic resistance cassette from plasmid pET28a (+), were reproduced and were connected to each other. The resulting fragment was cloned in specific position of pBluescriptIISK(-) plasmid by the restriction enzymes. Then, the construction was transferred into the genome of Brucella abortus by electroporation method. Results: Fusion PCR product was obtained without any change in the nucleotide sequence and then it was cloned into pBluescriptIISK (-) plasmid, finally the construction was replaced and the target gene was deleted. Conclusions: The results of this study show that the Overlap Extension PCR is an optimized and modified technique to create mutations in the bacterial genome structure and can easily be used in the family Brucella.

کلیدواژه‌ها [English]

  • Brucella abortus
  • mutation
  • overlap extension PCR
Anton, V.B., Matsumura, I. (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques. 48: 463-465.

Bikard, D., Jiang, W., Samai, P. (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41: 7429-7437.

Brian, M.F., Bitar, A.P. (2011) The metalloprotease of Listeria monocytogenes is regulated by pH. J Bacteriol. 193: 5090-5097.

Briones, G. (2001) Brucella abortus cyclic b-1, 2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells. Infect Immun. 69: 4528-4535.

Chou, C., Olszewski, N.E. (2004) Generation of random, in-frame mutations by TN1000-mediated mutagenesis. J Genet Mol Biol. 2: 137-142.

Denamur, E., Matic, I. (2006) Evolution of mutation rates in bacteria. Mol Microbial. 60: 820-7.

Eskra, L., Canavessi, A. (2001) Brucella abortus Genes Identified following Constitutive Growth and Macrophage Infection. Infect Immun. 69: 7736-7742.

Higuchi, R., Krummel, B. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16: 7351-67.

Horton, R.M. (1995) PCR-mediated recombination and mutagenesis. SOEing together tailor-made genes. Mol Biotechnol. 3: 93-9.

Horton, R.M., Cai, Z.L. (1990) Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques. 8: 528-35.

Jiang, W., Bikard, D. (2013) RNA-guided editing of bacterial genomes using crisPr-cas systems. Nat Biotechnol. 31: 233-9.

Kahl-McDonagh, M.M. (2006) Evaluation of protection afforded by Brucella abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates of clearance in BALB/c mice. Infect Immun. 74: 4048-4057.

Mariana, N.X., Tatiane, A.P. (2010) Pathogenesis of Brucella spp. Vet Sci J. 4: 109-118.

Mohamed, N.S., Stephen, M.B., Sriranganathan, N. (2008) Brucella: A pathogen without classic virulence genes. Vet Microbiol. 129: 1-14.

Monreal, D., Grillo, M.J. (2003) Characterization of Brucella abortus O-Polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model. Infect Immun. 71: 3261-71.

Nahid, S., Haghkhah, M. (2011) An efficient method for gene disruption in Brucella abortus by overlap extension PCR. Asian J Biothechnol. 3: 275-279.

Nicoletti, P. (2010) Brucellosis: past, present and future. Biol Med. 31: 21-32.

Oswald, R.C., Folkerts, O., Fei, Z. (2008) Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes. PLOS. 3: e2193.

Poester, F.P., Samartino, L.E., Santos, R.L. (2013) Pathogenesis and pathobiology of brucellosis in livestock. Rev Sci Tech. 32: 105-115.

Priscilla, C.H., Rene, M.T. (2000) Identification of genes required for chronic persistence of Brucella abortus in mice. Infect Immun. 68: 4102-7.

Ragan, V.E. (2002) The animal and plant health inspection service (APHIS) brucellosis eradication program in the United States. Vet microbiol. 90: 11-8.

Saeedinia, A.R., Zeinoddini, M., Soleimani, M. (2013) Deletion of perosamine synthetase gene in Brucella melitensis Rev1 to generate the attenuated mutant strain. J Police Med. 2: 127-138.

Sambrook, J., Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. (3rd ed.) CSHL Press. New York, USA.

Wallach, J.C., Ferrero, M.C., Victoria, M.D. (2008) Occupational infection due to Brucella abortus S19 among workers involved in vaccine production in Argentina. Clin Microbiol Infect. 14: 805-7.