Cloning and expression of Eimeria necatrix microneme5 gene in Escherichia coli

Document Type : Vaccinology

Authors

1 Faculty of Veterinary Medicine of Ahvaz

2 Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran

3 He is working at Veterinary Department of Qum State

4 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran

5 3Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran

Abstract

Background: Coccidiosis 
caused by Eimeria necatrix has the most economic impact on
poultry production. Micronemal proteins in Eimeria necatrix are thought
to be critical ligands determining host cell specificity at the time of
invasion.               OBJECTIVES: Isolation and purification of Eimeria necatrix oocysts from  Khuzestan province of Iran was performed. A
cDNA encoding microneme 5 (EnMIC5) protein was cloned and expressed as
recombinant protein before the evaluation of its immunogenicity by Western
blotting. METHODS: A primer pair was designed based on the published
nucleotide sequence of Eimeria necatrix LZ strain micronem5 gene. A
Partial cDNA sequence fragment of 758 bp coding for microneme 5 protein
(EnMIC5) was amplified by semi- Nested RT-PCR. PCR products were cloned and
expressed in a Maltose Binding protein (MBP) containing expression vector
(pMAL-c2x) in Escherichia coli. The cDNA which is encoded for 252 amino
acids shows high degree of conservation. It contains the adhesive plasma
pre-kallikrein and seven hydrophilic motifs. RESULTS: The results of SDS-PAGE
revealed that the recombinant protein with a molecular weight of 70 kDa was
over-expressed after induction with IPTG. Western blotting results revealed
that the expressed recombinant protein was reacted with sera of the chicks
infected with Eimeria necatrix. It was suggested that this protein
should have a good immunogenicity and can be used for further studies. CONCLUSIONS: In conclusion, the high degree of sequence homology indicates that this
protein is immunogenic and  might be an
interesting vaccine target, and deserves further investigation

Keywords

Article Title [Persian]

کلونینگ و بیان ژن میکرونم 5 ایمریا نکاتریکس در اشرشیاکلی

Authors [Persian]

  • منصور میاحی 1
  • عباس جلودار 2
  • شهروز مسائلی 3
  • حسین حمیدی نجات 4
  • مسعود صیفی آباد شاپوری 5
  • نغمه موری بختیاری 4
1 دانشکده دامپزشکی دانشگاه شهید چمران اهواز
2 گروه علوم پایه، بخش بیوشیمی و بیوتکنولوژی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز، ایران
3 در اداره کل دامپزشکی قم مشغول به مار است.
4 گروه پاتوبیولوژی، بخش انگل شناسی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز، ایران
5 گروه پاتوبیولوژی، بخش میکروب شناسی و ایمنی شناسی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز، ایران
Abstract [Persian]

زمینه مطالعه:  آیمریا نکاتریکس عامل کوکسیدیوز حداکثر تأثیر اقتصادی را بر روی تولید پرندگان دارد. به نظر می‌رسد پروتئین‌های میکرونم  نقش مهمی در حرکت انگل و تهاجم به سلول‌های میزبان دارد. هدف: جداسازی و خالص‌سازی اووسیست آیمریا نکاتریکس از استان خوزستان-ایران بود. یک cDNA کد کننده پروتئین میکرونم 5 (EnMIC5) کلون و پس از بیان کردن این پروتئین نوترکیب، خاصیت ایمنی‌زایی آن توسط وسترن بلوتینگ مورد آزمایش قرار گرفت. روش کار: یک جفت پرایمر بر اساس توالی نوکلئوتیدی موجود برای ژن میکرونم 5 آیمریا نکاتریکس سویه LZ طراحی و به وسیله آن توالی بخشی از cDNA کد کننده این پروتئین به میزان bp 758 با استفاده از روش Semi- Nested RT-PCR تکثیر گردید سپس قطعه بدست آمده به درون وکتور pMAL-c2x حاوی پروتئین باند شونده به مالتوز کلون و در باکتری E . coli سویه TG1 بیان گردید. قطعه تکثیر شده، یک open reading frame 252 اسیدآمینه ای داشت که به میزان قابل توجهی حاوی مناطق حفاظت شده شامل پری کالکرئین پلاسمایی چسبنده و هفت جزء آبدوست بود. نتایج: در SDS-PAGE پروتئین الحاقی با وزن مولکولی در حدود 70 کیلو دالتون پس از القاء توسط IPTG بیان شد. آزمایش وسترن بلات نشان داد که پروتئین نوترکیب با سرم بدست آمده از جوجه‌های آلوده به آیمریا نکاتریکس واکنش می‌دهد. نتیجه گیری نهایی: یافته‌ها نشان داد پروتئین بدست آمده قدرت ایمنی‌زایی ‌نسبتاً خوبی داشته و می‌تواند در آینده مورد مطالعه بیشتری قرار گیرد.

Keywords [Persian]

  • آیمریا نکاتریکس
  • بیان ژن
  • ایمنیزایی
  • میکرونم ______________________________________________

References

Bashar, A.E., Jian-ping, C., Ming-quan, X., Guo-qing, L., Zong-hua, Q., Hui-xian, W., Xing-yu, P., Weng-kang, W. (2003) Characterization of Microneme-2 (EtMlC-2) gene of Eimeia tenella Guangdong strain. Int J Poult Sci. 2: 133-138.
Bhogal, B., Miller, S., Anderson, G.A., Jessee, A.C., Strousberg, E.J., Mc Candliss, S., Nagle, R.J., Strousberg, R. (1992) Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervuline and E. tenella infections. Vet Immunol Immunopathol. 31: 323-335.
Blake, D.P., Shirley, M.W., Smith, A.L. (2006) Genetic identification of antigens protective against coccidia. Parasit Immunol. 28: 305-314.
Brown, P.J., Billington, K.J., Bumstead, J.M., Clark, J.D., Tomley, F.M. (2000) A microneme protein from Eimeria tenella with homology to the Apple domains of coagulation factor XI and plasma pre-kallikrein. J Molecul Biochem Parasitol. 107: 91-102.
Bumstead, J., Tomley, F.M. (2000) Induction of secretion and surface capping of microneme proteins in Eimeria tenella. J Molecul Biochem Parasitol. 110: 311-321.
Cai, X.P., Liu, H., Jia, W., Guo, A., Zhang, Sh., Yan, H., Yue, Ch. (2008) Recombinant expression of microneme protein 5 gene of Eimeria necatrix LZ strain in E. coli and antigenicity of recombinant protein. Act Vet Zootechnic Sini. 39: 1575 -1580.
Carruthers, V.B., Tomley, F.M. (2008) Receptor-ligand interaction and invasion: Microneme proteins in Apicomplexans. Subcell  Biochem. 47: 33-45.
Chomczynski, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analitic Biochem. 162: 156-159.
Crane, M.S.J., Goggin, B., Kirj, K.E., Chakoaboity, P.R. (1991) Cross protection against four species of chicken coccidian with a single recombinant antigen. Infect Immun. 59: 1271-1277.
Dubremetz, J.F. (1998) Apical organelles and host-cell invasion by Apicomplexa. Int J Parasitol. 28: 1007- 1013.
Jenkins, M.C. (1998) Progress on developing a recombinant coccidiosis vaccine. Int J Parasitol. 28: 1111-1119.
Jenkins, M.C. (2001) Advances and prospects for subunit vaccines against protozoa of veterinary importance. Vet Parasitol. 101: 291-310.
Johnston, D.A., Bratty, J., Fernando, M.A. (1998) A fast and inexpensive method for isolation of high-quality RNA from eimerian oocysts. Parasitol Res. 84: 549-551.
Kyte, J., Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J Molecul Biol. 157: 105-32.
Masaeli, Sh., Mayahi, M., Jolodar, A., Hamidinejat, H., Seyfi Abad Shapouri, M.R. (2011) Partial sequence determination of a cDNA encoding microneme 5 protein of Eimeria necatrix isolated in Khuzestan province, Iran. Iranian Journal of Veterinary Science Technology. 3: 39-48.
Murray, P.K., Bhogal, B.S., Crane, M.St J. (1986)  Eimeria tenella - in vivo immunization studies with sporozoite antigen. In: Research in Avian Coccidiosis. Mc Dougald, L.R., Joyner, L.P., Long, P.L. (eds.). University of Georgia Press. Athens, Georgia, USA. p. 564-573.
Qin, Z.H., Xie, M.Q., Cai, J.P., Elhag, A.B., Peng, X.Y., Wei, W.K., Wu, H.X. (2005) Expressing microneme-2 gene of Eimeria necatrix in Escherichia coli. Chin J Vet Sci. 25: 22-25.
Rose, M.E., Mockett, A.P.A. (1983) Antibodies to coccidia: detection by the enzyme-linked immunosorbent assay (ELISA). Parasite Immunol. 5: 479-489.
Shirley, M.W. (1995) Eimeria species and strains of chickens. In: Biotechnology Guidelines on Techniques in Coccidiosis Research. European Commission DGXII, Luxembourg. p. 1- 24.
Soldati, D., Brecht, S., Carruthers, V.B., Ferguson, D.J.P., Giddings, O.K., Wang, G., Jäkle, U., Harper, J.M., Sibley, L.D. (2001) The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains. J Biol Chem. 276: 4119-27.
Striepen, B., Soldati, D., Garcio-Reguet, N., Dubremetz, J.F., Roos, D. (2001) Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii. Molecul Biochem Parasitol. 113: 45- 53.
Su, Y.C., Fei, A.C., Tsai, F.M. (2003) Differential diagnosis of five avian Eimeria species by polymerase chain reaction using primers derived from the internal transcribed spacer 1 (ITS-1) sequence. Vet Parasitol. 117: 221-227.
Tomley, F.M. (1994) Characterization of rhoptry proteins of Eimeria tenella sporozoites: antigenic diversity of rhoptry epitopes within species of the genus Eimeria and among three asexual generations of a single species, E. tenella. Infect Immunol. 62: 4656-4658.
Tomley, F.M. (1997) Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites. Methods: A companion to Methods in enzymology. 13: 171-176.
Tomley, F.M., Clark, L.E., Kawazoe, U., Dijkema, R., Kok, J.J.  (1991) Sequence of gene encoding an immunodominant microneme protein of E. tenella. Molecul Biochem Parasitol. 49: 277-288.
Tomley, F.M., Billington, K.J., Bumstead, J.M., Clarka, J.D., Monaghanb, P. (1996) Molecular cloning and characterization of a novel acidic microneme protein (EtMIC-2) from the apicomplexan protozoan parasite, Eimeria tenella. Molecul Biochem Parasitol. 79: 195- 206.
Tomley, F.M., Billington, K.J., Bumstead, J.M., Clark, J.D., Monaghan, P. (2001) EtMIC-4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-1 repeats. Int J Parasitol. 31: 1303-1310.
Tomley, F.M., Soldati, D.S. (2001) Mix and match modules: structure and function of microneme protein in apicomplexan parasites. Parasitol Today. 31: 1303- 1310.
Vermeulen, A.N. (1998) Progress in recombinant vaccine development against coccidiosis. A review and prospects into the next millenium. Int J Parasitol. 28: 1121-1130.
Vermeulen, A.N., Kok, J.J., van den Boogart, P., Dijkema, R., Claessens, J.A.J. (1993) Eimeria refractile body proteins contain 2 potentially functional characteristice-transhydrogenase and carbohydrate transport. FEMS Microbiol Lett. 110: 223-230.
Williams, R.B. (1998) Epidemiological aspects of the use of live anticoccidial vaccines for chickens. Int J Parasitol. 28: 1089-1098.
Williams, R.B. (2006) Relative virulences of a drug-resistant and a drug-sensitive strain of Eimeria acervulina, a coccidium of chickens. Vet Parasitol. 15: 15-23.
Xie, Ming-quan., Qin, Zong-hua., Cai, Jian-ping., Bashar, A .E., Peng, Xin-yu., Wei, Wen-kang., Wu, Hui-xian. (2005) Immunogenic characterization of the recombinant attenuated Salmonella typhymurium expressing Mic2 gene of Eimeria necatrix. Acta Veterinaria et Zootechnica Sinica. 36: 705-710.
Iranian Journal of Veterinary Medicine
Volume 10, Issue 3
September 2016
Pages 157-163

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