An Egg Yolk Immunoglobulin (Rvp6-Igy) Specific for a Constructed
Rotavirus Vp6 Antigen (rVP6) Inhibited Rotavirus Replication in vitro
Marilen P. Balolong1,2*, Ju Kyoung Oh1, Jung Woo Kim1,
Yong Tae Jung3, Nina G. Gloriani4and Dae-Kyung Kang1
1School of Bio-resources Science, Dankook University
City of Cheonan 31116 Republic of Korea
2Department of Biology, College of Arts and Sciences,
University of the Philippines Manila, 1000 Philippines
3Department of Microbiology, Dankook University,
Dankook University, City of Cheonan 31116 Republic of Korea
4Department of Medical Microbiology, College of Public Health,
University of the Philippines Manila, 1000 Philippines
corresponding author:This email address is being protected from spambots. You need JavaScript enabled to view it.
ABSTRACT
Group A rotaviral diarrhea continues to be highly prevalent worldwide among children younger than 5 years of age, as well as among pre-weaning piglets. The middle capsid of rotavirus, VP6, is highly immunogenic and conserved among mammalian species, making it an ideal immunogen candidate. We developed a construct using the partial segment (nucleotides 8-1194) of the VP6 gene from Rotavirus strain OSU, subcloned into the expression vector pET 21b and expressed in Escherichia coli BL21 (DE3) to produce RVP6 that is ~45 kDa in size. Purification of RVP6 using a Ni-NTA column produced 3-4 mg L-1 of transformed E. coli culture after induction with 1 mM isopropyl beta-D-thiogalactoside (IPTG). RVP6 was then orally administered to mice to establish the characteristic immune response produced in serum and fecal samples. Likewise, RVP6 was also given intramuscularly to laying hens to recover RVP6-specific antibodies (RVP6-IgY) in yolk. RVP6-IgY was then tested for its ability to inhibit rotavirus replication in vitro. Three oral doses of RVP6 induced a characteristic systemic immune response as shown by increased serum IgG titer along with a complementary increase in fecal IgA titer suggestive of an induced mucosal response. It also mounted increased serum titers in laying hens, eventually recovering RVP6-IgY from yolk optimally at 6-weeks post immunization. The yolks with high titers were then selected for partial purification. Partially purified RVP6-IgY was shown to be specific to RVP6 immunogen (dot-blot assay) suggesting its potential for use in diagnostics. Replication was inhibited in vitro when RVP6-IgY was added before virus infection and when co-incubated with the virus at 100μg/ml concentration, suggesting its promise for prophylactic use. However, it was not able to inhibit replication when added post-infection. Our results provided basis to describe the potential of RVP6 and RVP6-IgY; therefore, efficacy studies in piglets are encouraged to confirm its potential.
INTRODUCTION
In order to gain food security for all, raising animals such as swine has been important, whether the rearing may be from a simple backyard farming practice or a commercialized husbandry. However, lapses during animal care practices, such as improper housing, feeding and management, can expose the pig population to several infectious disease-causing agents such as those causing viral gastroenteritis (Malik et al. 2014), particularly in developing countries. . . . . read more
