The Study Of Oral Immunization With The Transgenic Tomato Anti-caries Vaccine In Rabbits

Objective:Based on the third-generation seeds of the transgenic tomato which including interest gene pacA-ctxB or pacP-ctxB, to identificate the transgenic tomato and determinate of the interest protein by PCR, Western blot and ELISA.Further experiments of oral immunization in New Zealand rabbits were done in order to explore the immunizate effectiveness of the transgenic tomato expressed foreign protein PAcA/CTB,PAcP/CTB. Methods:The study included two parts.Test I:The detection of foreign gene pacA-ctxB or pacP-ctxB and interest protein in the transgenic tomato.1.To screening transgenic tomato plants which including the interest gene pacA-ctxB or pacP-ctxB,which encoding streptococcus mutans surface protein PAcA or PAcP and cholera toxin B subunit fusion by PCR.2. Determined total protein level by BCA.3.To determine the purpose protein expression by Western blot.4.Detected the concentration of interest protein by ELISA.Test II:The study of oral inmmunization with the transgenic tomato anti-caries vaccine in rabbits.1.Selection and grouping of animals:32 New Zealand white rabbits,divided into A, B,C,D 4 groups randomly. Group A divied to three dose groups.Group A1:Fed doses to 7ml/kg the transgenic tomato juice which expressed PAcA/CTB (including protein 53.3ug);Group A2:Fed doses to10ml/kg the transgenic tomato juice which expressed PAcA/CTB(containing the protein 76.20ug);Group A3:Fed doses to 13ml/kg the transgenic tomato juice which expressed PAcA/CTB(containing the protein 99.06ug).Group B which consists of three dose groups,too.Group B1:Fed doses to 7ml/kg the transgenic tomato juice which expressed PAcA/CTB (including protein 65.62ug);Group B2:Fed doses to10ml/kg the transgenic tomato juice which expressed PAcP/CTB (containing the protein 93.74ug); Group B3:Fed doses to 13ml/kg the transgenic tomato juice which expressed PAcP/CTB(containing the protein 121.87ug). Group C:Negative control group:Fed doses to10ml/kg untransgenic tomato juice.Group.D:Positive control group:Fed inactivated whole cell vaccine(once for 2ml).All aninals was assigned to 8 groups, and each group contained 4 animals.2. Immune ways:oral immunization.3. Immune time:once a week,four times.4. The time of sample collection:before the first immunizationand and after the immunization at 1,2,3,4,5,6,7,8,10,12 week respectively to collecting blood, saliva samples and weighed. 5. The detection of specific antibodies:To detect the IgG and IgA of serum and saliva by ELISA.6. Animals were sacrificed at 12 weeks, to take the heart, liver, spleen, lung, kidney for pathological section and observe the change of tissue and structure before and after immunity.Results:①With the total DNA as tempate of the transgenic tomato plants by the way of PCR and electrophoresis show 450bp and 506bp amplified bands,consistent with the positive control.②The total protein content were 4.644mg/ml and 3.456mg/ml By BCA method.segregate approximately about 60KD molecular protein strap banding. By SDS-PAGE and Western blot confirm the chimeric protein is PAcA/CTB or PAcP/CTB. Protein expression levels were 7.62μg/ml and 9.37μg/ml and accounted 0.19%and 0.27% in total protein respectively.③After oral immunizated the animals, the specific antibodies began to increase in rabbits'serum and saliva after 1 week, The levels of specific IgG antibodies and specific IgA antibodies in serum and saliva were significantly higher than the negative group(P<0.05), there was no different statistically significant between experimental group and the positive group, and there was no obvious significant difference between each dose group.④Each group rabbits were no significant differences in body weight at the same time(P>0.05).⑤Beside of inflammatory cells were detected in the positive control group, there were not obvious pathological change.Conclusions:①The transgenic tomato expressed the interest protein PAcA/CTB or PAcP/CTB successfully.These protein accounted for 0.16%and 0.27%in there total soluble protein respectively.②After immunited the rabbits with the trangenic tomato expressed frotein protein,I twere induced effectvely the antibodies anti-PAcA or anti-PAcP in salivary and blood of the rabbits.The transgenic tomato chimeric protein of PAcA/CTB and PAcP/CTB have better immunigenicity.