Cloning and expression of H gene of PPRV in baculovirus expression system

Subject Areas : Virology

Mehdi Taheri Asl ¹ , majid moghbeli ² , Mohammad Kargar ³ , mohsen lotfi ⁴ , Farshid Kafilzadeh ⁵

1 - Department of Microbiology, Islamic Azad University, Jahrom Branch, Jahrom, Iran
2 - Professor, Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.
3 - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
4 - Razi vaccine and serum research institute, agricultural research, education and extension organization(AREEO), Karaj, Iran.
5 - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran

Received: 2022-07-13 Accepted : 2022-11-05 Published : 2022-12-06

Keywords: Sf9 Cell, Recombinant, Peste des petits ruminants,

Abstract :

Background and Objectives: Small ruminant plague is one of the most economically important pathogens in sheep and goats caused by morbilli viruses. Protein H is one of the major proteins for immunity against PPRV. The aim of the present study was to clone and express the H gene of this virus by the baculovirus expression system.Materials and Methods: H gene was amplified by RT-PCR and specific primers and then cloned into pFastBac Dual plasmid. The recombinant vector with the gene was transferred to the DH10Bac host cell. By transfection Sf9 cell with recombinant vector, its expression and          characteristics were evaluated by SDS-PAGE, western blotting and Bradford methods.Results: The H gene was amplified using specific primers from the PPR virus genome and the   specific band was obtained. Cloning of the H gene on the pFastBac Dual vector and baculovirus genome was proved with PCR and enzymatic digestion. By transfection the recombinant vector into Sf9 cells and performing sequential passages, the recombinant protein was shown to be completely pure and specific using SDS-PAGE, Western blotting and Bradford methods. The     expression level of this gene was obtained 218 µg/ml.Conclusion: Considering the production of an appropriate amount of recombinant H protein, this protein can be a suitable candidate for the production of recombinant vaccine against PPRV.  

References:

Marashi M, Masoudi S, Moghadam MK, Modirrousta H, Marashi M, Parvizifar M, Dargi M, Saljooghian M, Homan F, Hoffmann B, Schulz C. Peste des petits ruminants virus in vulnerable wild small ruminants, Iran, 2014–2016. Emerging infectious diseases. 2017 Apr;23(4):704.

Woma TY, Adombi CM, Yu D, Qasim AM, Sabi AA, Maurice NA, Olaiya OD, Loitsch A, Bailey D, Shamaki D, Dundon WG. Co‐circulation of peste‐des‐petits‐ruminants virus Asian lineage IV with lineage II in Nigeria. Transboundary and emerging diseases. 2016 Jun;63(3):235-42.

Tounkara K, Kwiatek O, Niang M, Abou Kounta Sidibe C, Sery A, Dakouo M, Salami H, Lo MM, Ba A, Diop M, Mamy E. Genetic evidence for transboundary circulation of peste des petits ruminants across West Africa. Frontiers in veterinary science. 2019 Aug 21;6:275.

FAOSTAT Food and Agriculture Data of the Food and Agriculture Organization of the United Nations. (https://www.fao. org/faostat/en/#data/QA) Accessed June 2020

Kumar N, Barua S, Riyesh T, Tripathi BN. Advances in peste des petits ruminants vaccines. Veterinary microbiology. 2017 Jul 1;206:91-101.

Baazizi R, Mahapatra M, Clarke BD, Ait-Oudhia K, Khelef D, Parida S. Peste des petits ruminants (PPR): A neglected tropical disease in Maghreb region of North Africa and its threat to Europe. PloS one. 2017 Apr 20;12(4):e0175461.

Cotmore SF, Agbandje-McKenna M, Canuti M, Chiorini JA, Eis-Hubinger AM, Hughes J, Mietzsch M, Modha S, Ogliastro M, Pénzes JJ, Pintel DJ. ICTV virus taxonomy profile: Parvoviridae. The Journal of general virology. 2019 Mar;100(3):367.

Fakri F, Bamouh Z, Ghzal F, Baha W, Tadlaoui K, Fihri OF, Chen W, Bu Z, Elharrak M. Comparative evaluation of three capripoxvirus-vectored peste des petits ruminants vaccines. Virology. 2018 Jan 15;514:211-5.

Niyokwishimira A, de D Baziki J, Dundon WG, Nwankpa N, Njoroge C, Boussini H, Wamwayi H, Jaw B, Cattoli G, Nkundwanayo C, Ntakirutimana D. Detection and molecular characterization of Peste des Petits Ruminants virus from outbreaks in Burundi, December 2017–January 2018. Transboundary and emerging diseases. 2019 Sep;66(5):2067-73.

Zhou XY, Wang Y, Zhu J, Miao QH, Zhu LQ, Zhan SH, Wang GJ, Liu GQ. First report of peste des petits ruminants virus lineage II in Hydropotes inermis, China. Transboundary and emerging diseases. 2018 Feb;65(1):e205-9.

Schägger H. Tricine–sds-page. Nature protocols. 2006 Jun;1(1):16.

Zhou XY, Wang Y, Zhu J, Miao QH, Zhu LQ, Zhan SH, Wang GJ, Liu GQ. First report of peste des petits ruminants virus lineage II in Hydropotes inermis, China. Transboundary and emerging diseases. 2018 Feb;65(1):e205-9.

Patel JM, Patel DR, Mavadiya SV, Solanki JB, Vihol PD, Sharma KK, Trangadia BJ, Kalyani IH. Clinicopathological investigation of an outbreak of peste des petits ruminants in small ruminants in South Gujarat, India. Indian Journal of Veterinary Pathology. 2015;39(1):20-3.

Truong T, Boshra H, Embury-Hyatt C, Nfon C, Gerdts V, Tikoo S, Babiuk LA, Kara P, Chetty T, Mather A, Wallace DB. Peste des petits ruminants virus tissue tropism and pathogenesis in sheep and goats following experimental infection. PloS one. 2014 Jan 30;9(1):e87145.

Zhou XY, Wang Y, Zhu J, Miao QH, Zhu LQ, Zhan SH, Wang GJ, Liu GQ. First report of peste des petits ruminants virus lineage II in Hydropotes inermis, China. Transboundary and emerging diseases. 2018 Feb;65(1):e205-9.

Herbert, R., et al. (2014). "Recombinant adenovirus expressing the haemagglutinin of peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR." Veterinary research 45(1): 24.

Fakri F, Ghzal F, Daouam S, Elarkam A, Douieb L, Zouheir Y, Tadlaoui K, Fassi-Fihri O. Development and field application of a new combined vaccine against Peste des Petits Ruminants and Sheep Pox. Trials in vaccinology. 2015 Jan 1;4:33-7.

Liu F, Wu X, Zhao Y, Li L, Wang Z. Budding of peste des petits ruminants virus-like particles from insect cell membrane based on intracellular co-expression of peste des petits ruminants virus M, H and N proteins by recombinant baculoviruses. Journal of virological methods. 2014 Oct 1;207:78-85.

Apsana R, Balamurugan V, Veeregowda BM, Abraham S, Raju DS, Rathnamma D, Byregowda SM, Rahman H, Shaila MS. Expression and characterization of immunodominant region of fusion protein of Peste des petits ruminants virus in E. coli. Small Ruminant Research. 2016 Nov 1;144:75-82.