טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentPaz Harel Gili
SubjectExpression of the LT Gene in Plants for Increasing
Immunogenicity of Vaccines for Animals, Produced
in Transgenic Plants
DepartmentDepartment of Biology
Supervisors Professor Emeritus Shimon Gepstein
Dr. Eli Khayat


Abstract

Our research focuses on the development of transgenic plants that would express efficient vaccines for oral vaccination. Our aim was to develop edible vaccines whose role is to prime the mucosal immune system to swiftly destroy a specific disease.

Oral immunization through feed is a way of immunization saving time and labor and which avoids any handling stress. However, oral administration has been disappointing so far, providing generally weak protection.

One of the possible solutions is to use an effective biological adjuvant, E.coli Heat Labile Enterotoxin (LT - causing Travelers’ diarrhea).

Due to its toxicity, the use of WT LT as a stimulator is undesirable. Harmless versions of the polypeptide are developed. Mutated toxins are used, yet not in transgenic plants, in which the toxicity is lost while the adjuvant activity is retained. We have employed a mutant of LT, the LT (R192G). The mutation abolished the toxicity without affecting its immuno-stimulant activity.

As a first step towards developing the transgenic plant with the biological adjuvant, the LT (R192G) gene was inserted into a binary plasmid under a constitutive plant promoter (35S). Then, tobacco plants were transformed with the LT gene. For analyzing the efficiency of this protein and for comparison, the LT (R192G) gene was also expressed in E.Coli.

Transformed bacteria and transgenic plants were tested for the LT protein expression by SDS-PAGE and found ready for feeding experiments. Bacteria and plant protein crude extracts were fed to chicken, and the immune response was quantified by Western blot analysis. The LT protein-containing extracts were introduced into the tested chicken with the virus causing the Newcastle, a known chicken disease.

IgA and IgG levels in the chicken sera were measured. In both cases, antibody levels were higher when the adjuvant was included as compared to levels measured where the virus was introduced alone.

These results indicated that the plant-derived LT (R192G) indeed enhances the immunogenic response and may be considered as a potential biological adjuvant especially for oral administration of transgenic plants.

We demonstrated that transgenic tobacco plants expressing the LT (R192G) gene may be a potential source of oral vaccines for animals.