Sornkanok Vimolmangkang, Ph.D. Dept. Pharmacognosy and Pharmaceutical botany, Faculty of...
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Transcript of Sornkanok Vimolmangkang, Ph.D. Dept. Pharmacognosy and Pharmaceutical botany, Faculty of...
Sornkanok Vimolmangkang, Ph.D.Dept. Pharmacognosy and Pharmaceutical botany, Faculty
of Pharmaceutical sciences, Chulalongkorn University
Dept. Natural Resources and Environmental Sciences,
University of Illinois at Urbana-Champaign
Soybean-based viral subunit vaccine against porcine
reproductive and respiratory syndrome (PRRS)
Porcine Reproductive and Respiratory Syndrome
Deadly swine disease caused by PRRS virusEconomic losses in swine production
worldwide and up to 560 million US dollars (Neumann et al., 2005)
Impact of PRRS on farm Increased mortality (1.9 to 10.2 %) Reduced average daily gain (0.38 to 0.26
kg) Increased treatment cost per pig (US$ 1.77
to 1.91) Reduced feed efficiency (1.77 to 1.91 kg
feed per kg gain)
Dee S. et al., 1997
http://www.thepigsite.com/pighealth/article/142/porcine-reproductive-and-respiratory-syndrome-prrs
Clinical signsNo symptoms to
death observed in pregnant sows, piglets, and gilts
Respiratory distress observed in all ages
Reproductive distress leads to premature farrowing or abortion in pregnant sows
Stillborn piglet
Mummified PRRS piglet
PRRS control strategiesInnovative diagnostic toolsFarm management strategies
Temperature control Strict sanitation
Effective PRRS vaccineModified live vaccine Viral subunit vaccine
Despite current vaccines are available in the market, it remains unsatisfied by producer needs.
The development of an effective PRRS vaccine that is reliable, easy to administer, and safe remains a major challenge.
an alternative vaccination strategy by using plant production systems for antigenic proteins
Benefits of plant-based vaccine InexpensiveSafeScalableParenteral or oral administration (orally
used without purification)Systemic and mucosal immune responses
Plant-based oral vaccine against swine diseases
porcine epidemic diarrhea virus (PEDV) (Kang et al., 2005; Oszvald et al., 2007)
porcine transmissible gastroenteritis virus (Streatfield et al., 2001; Lamphear et al., 2002 )
PRRSV (Chia et al., 2010; 2011; Chen and Lu, 2011)
ObjectiveTo transfer and generate transgenic
soybean carrying candidate PRRS viral subunit genes by using Agrobacterium-mediated transformation
Development of plant-based vaccine
Select a targetIdentify a coding sequence
Agrobacterium-mediated transformation
Somatic embryogenesis
SEs
Transfer Transformation
Regeneration
Transgenic soybean
Animal feeding studies
PRRS viral structure1 nt 15428 nt
Non structural protein region Structural protein region
ORF1a
1b2
34
56
7
Single-stranded RNA
15 kDa N protein (ORF 7 product)
30 - 40 kDa GP4 (ORF 4 product)
18 - 19 kDa M protein (ORF 6 product)
24 -26 kDa GP5 (ORF 5 product)
Immunogenicity
Structural proteins encoded from ORF 2 to 7 were tested in pigs and found their ability to induce immune responses
The product of ORF6 contributed to the highest response, stimulated cellular immunity (Bautista et al., 1999)
Neutralizing antibodies were detected with the highest titres in ORF5 vaccinated pigs, the consistent induction of antibodies were observed in ORF7 vaccinated pigs after three vaccinations (Barfoed et al., 2004).
Plasmid construction
Intermediate vector
Expression vector
Soybean host systemAn ideal system for testing the concept of
“value-added crops” Protein-rich animal feed Easily transported worldwide in large
quantities with no special care Ability to be transformed
Soybean transformation
Transgene expression
8 putative transgenic T0 lines 5 from Jack (Ga1, Ga2, Ga3, Ha1, Ha2)3 from Kunitz (Oa1, Pa1, Pa2)
Jack: 1/5 PCR-positive (Ga1)Kunitz: 3/3 PCR-positive (Oa1, Pa1, Pa2)
Southern blot analysis for transgene integration and T-DNA copy number
RT-PCR showed the presence of mRNA with different level of expression among transgenic events
Ga1 ~ Oa1 Pa1 ~ Pa2Ga1,Oa1 > Pa1,
Pa2
a. PRRSV-ORF7 primers; b. soybean tubulin primers (internal control)
The amount of the antigenic protein produced in transgenic lines was determined using ELISA
Kunitz
Jack
ConclusionFour transgenic plants were developed: one
individual from cv. Jack and three from cv. Kunitz.
All plants regenerated from Kunitz were transformed.
The presence of transgene DNA, mRNA, and protein prove the concept that PRRS viral subunit can be expressed in soybean
Future worksExpression of other PRRS viral subunits in
soybean Generation of homozygous transgenic linesAntigenicity assessment of these candidate
oral plant-based vaccines in mice and swine
AcknowledgementDr. Schuyler S. Korban Dr. Ksenija GasicDr. Yuepeng HanLab-matesRoyal Thai Government