ترانسفکت سلول حشره Sf9 توسط باکولوویروس نوترکیب واجد ژن Core+1 ویروس هپاتیت سی سویهJFH1 ژنوتایپ 2a به منظور بیان این پروتئین
محورهای موضوعی : زیست فناوری میکروبیفریده سادات سجادیان فرد 1 , پونه رحیمی 2
1 - کارشناس ارشد، گروه میکروب شناسی، دانشکده علوم و فناوری های نوین، واحد علوم دارویی دانشگاه آزاد اسلامی-تهران
2 - دانشیار، بخش هپاتیت و ایدز، انستیتو پاستور ایران، گروه ویروس شناسی پزشکی، تهران
کلید واژه: پروتئین Core+1, ویروس هپاتیت سی, سویه JFH1, سلول حشره Sf9, سیستم بیانی باکولوویروس,
چکیده مقاله :
سابقه و هدف: در سال های اخیر پروتئین جدیدی بنام Core+1 گزارش شده است که به وسیله یک تغییر ریبوزومی 1+ در ناحیه کد کننده پروتئین Core ویروس هپاتیت سی تولید می شود. این مطالعه با هدف طراحی و ساخت وکتور باکولوویروس واجد توالی Core+1 HCV-2a (JFH1) تولید باکولوویروس نوترکیب و تایید ترانسفکت آن در سلول های حشره انجام شد. مواد و روش ها: در این مطالعه، از ژن Core+1 HCV-2a (JFH1) که در پلاسمید pUC57 به صورت تجاری سنتز شده بود استفاده گردید. ژن مورد نظر در پلاسمید pFastBac-HTA همسانه سازی مجدد شد و توسط این وکتور انتقالی در میزبان اشریشیا کلی DH10Bac با عمل ترانسپوزیشن، بکمید باکولوویروس نوترکیب به دست آمد. سازه نوترکیب پس از تایید با واکنش زنجیره ای پلی مراز، برای ساخت با کولوویروس نوترکیب در سلول حشره Sf9 ترانسفکت گردید. وجود پروتئین نوترکیب Core+1 در سلول حشره با روش SDS-PAGE و وسترن بلات مورد تایید قرار گرفت. یافته ها: همسانه سازی صحیح ژن 1+Core در وکتور انتقالی pFastBac با استفاده از برش آنزیمی و تعیین توالی تایید گردید. واکنش زنجیره ای پلی مراز نشان دهنده صحت طول نواحی تکثیر یافته هدف روی بکمید، ایجاد ترانسپوزیشن و نوترکیبی بکمید نوترکیب بود. اثرات سایتوپاتیک سلول Sf9 نشان دهنده ایجاد باکولوویروس نوترکیب بود. پروتئینCore+1 HCV-2a (JFH1) به طور موفقیت آمیز بیان گردید. نتیجه گیری: با طراحی و ساخت وکتور باکولوویروس و تولید باکولوویروس نوترکیب می توان پروتئین Core+1 مشابه با پروتئین ساخته شده ویروسی را بیان نمود. این عمل پایه انجام مطالعات آینده خواهد بود.
Background & Objectives: Recently, a new protein, named Core+1, has been reported to be expressed through a +1 ribosomal frame shift in the Core protein coding region of Hepatitis C virus. The purpose of this study was to design a recombinant Baculovirus vector containing HCV-2a (JFH1) Core+1 sequence, and also, to verify the production of this recombinant Core+1 protein in Baculovirus expression system. Materials & Methods: Core+1 gene of HCV-2a (JFH1) was synthesized into pUC57 plasmid. The synthesized target gene was sub-cloned into the plasmid pFastBac-HTA. The recombinant vector was used to transform the competent E. coli DH10Bac containing the donor clone. The recombinant Baculovirus bacmid was produced following transposition. Recombinant bacmid was verified by PCR and then was transfected into Sf9 insect cells to package a new recombinant Bbaculovirus. SDS-PAGE and Western blotting was used to confirm the expression of Core+1 protein in insect cells. Results: Sequence analysis and white-blue colony selection confirmed a successful cloning of the Core+1 sequence of HCV (JFH1) into the pFastBac-HTA vector and transformation of E. coli DH10Bac. Production of recombinant Baculovirus. The HCV-2a (JFH1) Core+1 protein was successfully expressed and confirmed in SDS-PAGE and Western blot. Conclusions: Baculovirus expression system provides a high yield post-translational modification tool for HCV Core+1 protein expression, which is similar to Core+1 protein produced during natural infection with HCV.
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2. Khatibi M, Ahmadinejad Z, Nasiri-Toosi M, Hajibaygi B, Zahedipour H. Prevalence of oral lichen planus in HCV infected patients: the effective factors. Teh Univ Med J. 2008; 66(8): 585-589. [In Persian]
3. Vassilaki N, Mavromara P. The HCV ARFP/F/core+1 protein: production and functional analysis of an unconventional viral product. IUBLB life. 2009; 61(7): 739-752.
4. Jones AM, Warken K, Tyring Sk. The cutaneous manifestations of viral hepatitis. Dermatol Clin. 2002; 20(2): 233-247.
5. Joyce MA, Tyrrell DLJ. The cell biology of hepatitis C virus. Microbes Infect. 2010; 12(4): 263-271.
6. Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS, Pybus OG, Barnes, E. Global distribution and prevalence of hepatitis C virus genotypes. Hepatol. 2015; 61(1): 77-87.
7. Donald Jensen, Nancy Reau. Hepatitis C. Oxford American Infectious Disease Library; 2013: 15-21. Available from: https://books.google.com/books.
8. Aghasadeghi MR, Sadat SM, Amini S, Budkowska A, Roohvand F. Cloning, optimization of expression condition, purification and immunological characterization of hydrophilic section of HCV Core Ag, expressed in E. coli by -araBAD promotor. SJIBTO. 2006; 2(6): 223-231. [In Persian]
9. Noorbazargan H, Hashemi A, Motavali F, Aghasadeghi MR, Memarnejadian A, Assmar M, Roohvand F. A confocal-microscopic study on HCV core+1 protein expression. J Microb World. 2010; 2(4): 217-226. [In Persian]
10. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige k, Mizokami M, Wakita T. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon .Gastroenterol. 2003; 125 (6): 1808-1817.
11. Kato T, Furusaka A, Miyamoto M, Date T, Yasui K, Hiramoto J, Nagayama K, Tanaka T, Wakita T. Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol. 2001; 64(3): 334-339.
12. Brown TA. Gene cloning and DNA analysis. 6nd ed. Translated by Tabatabi yazdi M, Zarrini GH, Sepehri zadeh Z, Ghasemian A, Hemat A. Tehran. Biology home publication; 2010. [In Persian]
13. Wang X, Li L, Ding S, Huang X, Zhang J, Yin J, Zhong J. Chicken HS4 insulator significantly improves baculovirus-mediated foreign gene expression in insect cells by modifying the structure of neighbouring chromatin in virus mini-chromosome. J Biotechnol. 2009; 142(3-4): 193-199.
14. Guide to Baculovirus expression vector systems (BEVS) and insect cell culture techniques. Invitrogen by life technologies. Available from: https://tools.lifetechnologies.com/content/sfs/manuals/ bevtest.pdf
15. Murphy CI, Piwnica-Worms H, Grünwald S, Romanow WG, Francis N, Fan HY. Expression and purification of recombinant proteins using the Baculovirus system. Curr Protoc Mol Biol. 2004; 16: 11.
16. Patterson RM, Selkirk JK, Merrick BA. Baculovirus and insect cell gene expression: review of baculovirus biotechnology. Environ Health Perspect. 1995; 103(7-8): 756-759.
17. Hervas-Stubbs S, Rueda P, Lopez L, Leclerc C. Insect Baculoviruses strongly potentiate adaptive immune responses by inducing type I IFN. J Immunol. 2007; 178(4): 2361-2369.
18. Addgene. Available from: https://www.addgene.org.
19. Bac-to-Bac® Baculovirus expression system. Invitrogen by life technologies. Available from: https://tools.thermofisher.com/content/sfs/manuals/bactobac_man.pdf.
20. Rabiei M, Mohtasham Amiri Z. Prevalence of Lichen planus in HCV infected patients of Gilan province 2002. Shahid Beheshti Med Sci Univ J Dental School. 2003; 21(2): 193-200.
21. Ray RB, Meyer K, Steele R, Shrivastava A, Aggarwal BB, Ray R. Inhibition of tumor necrosis factor (TNF- α)- mediated apoptosis by Hepatitis C virus core protein. J Biol Chem. 1998; 273(4): 2256-2259.
22. Baghbani-arani F, Roohvand F, Aghasadeghi MR, Sadat S M, Motevalli F, Memarnejadian, Amini S. Study the capacity of recombinant HCV Core+1 protein to induce immune responses in combination with different adjuvants. J Army Univ Med Sci. 2012; 10(1): 1-9. [In Persian]
23. Behzadian F, Goodarzi Z, Saberfar E. Construction of a new recombinant Baculovirus encoding HA, Na, and M1 proteins of swine influenza (H1N1) virus and its expression in insect cells. AMUJ. 2013; 15(67): 16-25. [In Persian]
24. Hirowatari Y, Hijikata M, Tanji Y, Shimotohno K. Expression and processing of putative nonstructural proteins of Hepatitis C virus in insect cells using Baculovirus vector. Virus Res. 1995; 35(1): 43-61.
25. Rodríguez-Rodrígueza M, Tello D, Yélamos B, Gomez-Gutierrez J, Pacheco B, Ortega S, Serrano A G, Peterson D L, Gavilanes F. Structural properties of the ectodomain of Hepatitis C virus E2 envelope protein. Virus Res. 2009; 139(1): 91-99.
26. Hussy P, Faust H, Wagner J-C, Schmid G, Mous J, Jacobsen H. Evaluation of Hepatitis C virus envelope proteins expressed in E. coli and insect cells for use as tools for antibody screening. J Hepatol. 1997; 26(6): 1179-1186.
27. Ciccaglione AR, Marcantonio C, Equestre M, Jones IM, Rapicetta M. Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the Baculovirus insect cell system. Virus Res. 1998; 55(2): 157-165.
_||_1. Assarehzadegan MA, Shakerinejad Gh, Norouzirad R, Amini A. Distribution of hepatitis C virus genotypes among patients with hepatitis C infection in Khuzestan province. J Med Sci. 2009; 7(4): 471-478. [In Persian]
2. Khatibi M, Ahmadinejad Z, Nasiri-Toosi M, Hajibaygi B, Zahedipour H. Prevalence of oral lichen planus in HCV infected patients: the effective factors. Teh Univ Med J. 2008; 66(8): 585-589. [In Persian]
3. Vassilaki N, Mavromara P. The HCV ARFP/F/core+1 protein: production and functional analysis of an unconventional viral product. IUBLB life. 2009; 61(7): 739-752.
4. Jones AM, Warken K, Tyring Sk. The cutaneous manifestations of viral hepatitis. Dermatol Clin. 2002; 20(2): 233-247.
5. Joyce MA, Tyrrell DLJ. The cell biology of hepatitis C virus. Microbes Infect. 2010; 12(4): 263-271.
6. Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS, Pybus OG, Barnes, E. Global distribution and prevalence of hepatitis C virus genotypes. Hepatol. 2015; 61(1): 77-87.
7. Donald Jensen, Nancy Reau. Hepatitis C. Oxford American Infectious Disease Library; 2013: 15-21. Available from: https://books.google.com/books.
8. Aghasadeghi MR, Sadat SM, Amini S, Budkowska A, Roohvand F. Cloning, optimization of expression condition, purification and immunological characterization of hydrophilic section of HCV Core Ag, expressed in E. coli by -araBAD promotor. SJIBTO. 2006; 2(6): 223-231. [In Persian]
9. Noorbazargan H, Hashemi A, Motavali F, Aghasadeghi MR, Memarnejadian A, Assmar M, Roohvand F. A confocal-microscopic study on HCV core+1 protein expression. J Microb World. 2010; 2(4): 217-226. [In Persian]
10. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige k, Mizokami M, Wakita T. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon .Gastroenterol. 2003; 125 (6): 1808-1817.
11. Kato T, Furusaka A, Miyamoto M, Date T, Yasui K, Hiramoto J, Nagayama K, Tanaka T, Wakita T. Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol. 2001; 64(3): 334-339.
12. Brown TA. Gene cloning and DNA analysis. 6nd ed. Translated by Tabatabi yazdi M, Zarrini GH, Sepehri zadeh Z, Ghasemian A, Hemat A. Tehran. Biology home publication; 2010. [In Persian]
13. Wang X, Li L, Ding S, Huang X, Zhang J, Yin J, Zhong J. Chicken HS4 insulator significantly improves baculovirus-mediated foreign gene expression in insect cells by modifying the structure of neighbouring chromatin in virus mini-chromosome. J Biotechnol. 2009; 142(3-4): 193-199.
14. Guide to Baculovirus expression vector systems (BEVS) and insect cell culture techniques. Invitrogen by life technologies. Available from: https://tools.lifetechnologies.com/content/sfs/manuals/ bevtest.pdf
15. Murphy CI, Piwnica-Worms H, Grünwald S, Romanow WG, Francis N, Fan HY. Expression and purification of recombinant proteins using the Baculovirus system. Curr Protoc Mol Biol. 2004; 16: 11.
16. Patterson RM, Selkirk JK, Merrick BA. Baculovirus and insect cell gene expression: review of baculovirus biotechnology. Environ Health Perspect. 1995; 103(7-8): 756-759.
17. Hervas-Stubbs S, Rueda P, Lopez L, Leclerc C. Insect Baculoviruses strongly potentiate adaptive immune responses by inducing type I IFN. J Immunol. 2007; 178(4): 2361-2369.
18. Addgene. Available from: https://www.addgene.org.
19. Bac-to-Bac® Baculovirus expression system. Invitrogen by life technologies. Available from: https://tools.thermofisher.com/content/sfs/manuals/bactobac_man.pdf.
20. Rabiei M, Mohtasham Amiri Z. Prevalence of Lichen planus in HCV infected patients of Gilan province 2002. Shahid Beheshti Med Sci Univ J Dental School. 2003; 21(2): 193-200.
21. Ray RB, Meyer K, Steele R, Shrivastava A, Aggarwal BB, Ray R. Inhibition of tumor necrosis factor (TNF- α)- mediated apoptosis by Hepatitis C virus core protein. J Biol Chem. 1998; 273(4): 2256-2259.
22. Baghbani-arani F, Roohvand F, Aghasadeghi MR, Sadat S M, Motevalli F, Memarnejadian, Amini S. Study the capacity of recombinant HCV Core+1 protein to induce immune responses in combination with different adjuvants. J Army Univ Med Sci. 2012; 10(1): 1-9. [In Persian]
23. Behzadian F, Goodarzi Z, Saberfar E. Construction of a new recombinant Baculovirus encoding HA, Na, and M1 proteins of swine influenza (H1N1) virus and its expression in insect cells. AMUJ. 2013; 15(67): 16-25. [In Persian]
24. Hirowatari Y, Hijikata M, Tanji Y, Shimotohno K. Expression and processing of putative nonstructural proteins of Hepatitis C virus in insect cells using Baculovirus vector. Virus Res. 1995; 35(1): 43-61.
25. Rodríguez-Rodrígueza M, Tello D, Yélamos B, Gomez-Gutierrez J, Pacheco B, Ortega S, Serrano A G, Peterson D L, Gavilanes F. Structural properties of the ectodomain of Hepatitis C virus E2 envelope protein. Virus Res. 2009; 139(1): 91-99.
26. Hussy P, Faust H, Wagner J-C, Schmid G, Mous J, Jacobsen H. Evaluation of Hepatitis C virus envelope proteins expressed in E. coli and insect cells for use as tools for antibody screening. J Hepatol. 1997; 26(6): 1179-1186.
27. Ciccaglione AR, Marcantonio C, Equestre M, Jones IM, Rapicetta M. Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the Baculovirus insect cell system. Virus Res. 1998; 55(2): 157-165.