The Prelliminary Study Of The Safety On The Transgenic Tomato Anti-caries Vaccine

Objective:To detect the exogenous gene pacA-ctxB,pacP-ctxB and the expression of target protein PAcA/CTB,PAcP/CTB in the transgenic tomatoes and non-transgenic plants, and through observing the toxicity of the transgenic tomatoes in the immunized animals to investigate the ecological safety of transgenic plant anti-caries vaccine. Methods:This study consists of three partsTest 1:Detection of transgenic tomato:To sieve transgenic plants carrying exogenous gens pacA-ctxB,pacP-ctxB from transgenic tomato by PCR, detect the expression of foreign protein by Western blot, and to determine exogenous interest protein's content by ELISA. Test 2:Ecological safety of transgenic tomato.1. Detection of exogenous gene drifted with pollen:Plant the transgenic tomatoes in the field, with non-transgenic tomato growing separately away from 0.5m, 1m, 3m and 10m as drift acceptor, all tomatoes was pollinated random in the efflorescence. Till the time of fructification, tomato fruits were collected from 10 plants stochastically and cultivated in the lab. Finally the blades of seeding stage were detected by PCR.2. Detection of rhizosphere edaphic: to detect edaphic DNA from soil samples from four directions of tomato roots by PCR.3. Detection of copy number of the transgenic tomato gene:to take recombinant plasmid pEAC10 and pEPC10 for standard,and detect genome samples of exogenous gene pacA-ctxB and pacP-ctxB by SYBR Green fluorescent quantitation, and take the average as gene copy number.Test 3:The safety of animals with the transgenic tomatoes.1. The study of acute toxicity in rats:(1) Animal selection and grouping:24 wistar rats with half male, divided into 3 groups randomly. Group A:the transgenic tomato juice immunity class with PAcA/CTB protein; Group B:the transgenic tomato juice immunity class with PAcP/CTB protein; Control group C:the non-transgenic tomato juice immunity class.(2)Immunization scheme:20g/kg of tomato juice was given 3 times per day.(3)observed index:①weigh animals every other day.②record the time when symptom of toxic reaction appeared and disappeared, and record the death time as well③sacrifice all animals on the seventh day and take heart, liver, kidney, spleen and thymus for histopathology inspection.2.The subchronic toxicity study of rats:(1) Animal selection and grouping:72 wistar rats with half males, divided into 6 groups randomly. Group A1:20g/kg the transgenic tomato juice immunity class with PAcA/CTB protein. Group A2:10g/kg the transgenic tomato juice immunity class with PAcA/CTB protein. Group B1:20g/kg the transgenic tomato juice immunity class with PAcA/CTB protein. Group B2:10g/kg the transgenic tomato juice immunity class with PAcA/CTB protein. Group C1:20g/kg the non-transgenic tomato juice immunity class. Group C2:10g/kg the non-transgenic tomato juice immunity class.(2)Immunization scheme:continuous intragastric administration for 30 days.(3)observed index:①weigh the animal every day during the experiment.②observe the activities and appearances of animals every day.③do the hematology, blood biochemistry and urine examination after 30-day lavage④take heart, liver, kidney, spleen and thymus to weigh for organ coefficient calculation.⑤take heart, liver,kidney, spleen and thymus for histopathology inspection.3. Study of mutagenic test3.1 Bone marrow micronucleus test:(1)Animal selection and grouping:42 Kunming mice with half male, divided into 7 groups randomly. Group A1:5g/kg the transgenic tomato juice immunity class with PAcA/CTB protein. Group A2:2g/kg the transgenic tomato juice immunity class with PAcA/CTB protein. Group B1:5g/kg the transgenic tomato juice immunity class with PAcA/CTB protein. Group B2:2g/kg the transgenic tomato juice immunity class with PAcA/CTB protein. Negative control group C1:5g/kg the non-transgenic tomato juice immunity class. Negative control group C2:2g/kg the non-transgenic tomato juice immunity class. Positive control group D for positive control:100mg/(kg*d) subcutaneous injection of cyclophosphamide; (2) Immunization scheme:lavage to test and positive control group for 4 days, and animals were injected cyclophosphamide i.p for 4 days and sacrificed on 5th days.(3)observed index:investigate micronuclear rates of the micronuclei polychromatic cells with a microscope in the mouse bone marrow films.3.2 Sperm abnormality test:(1)Animal selection and grouping:42 Kunming mice, divided into 7 groups randomly, Positive control group D was selected as positive control:20mg/(kg*d) subcutaneous injection of cyclophosphamide, other groups as the same as bone marrow micronucleus test. (2)Immunization scheme:lavage for test and positive control group for 5 days, and animals were injected cyclophosphamide i.p for 5 days and sacrificed on 35th days(3)observed index:prepare sperm films, and investigate teratospermia ratio of the teratospermia cells with a microscope averagely.4. Generation reproductive toxicity test(1) Animal selection and grouping:60 SD rats with half male, divided into 3 groups randomly. Group A:the transgenic tomato juice immunity class with PAcA/CTB protein; Group B:the transgenic tomato juice immunity class with PAcP/CTB protein; Control group C: non-transgenic tomato juice immunity class.(2)Immunization scheme:after male rats have been administrated intragastrically for 8 weeks, females started the intragastric administration.2 weeks later, male and female rats were mated 1:1 into the same cage with continuous intragastric administration.3 more weeks later, the males were sacrificed and the intragastric administration of females were continued till offspring weaning for 3 weeks.(3)observed index:①observe the rats once a day and weight the mating rats once a week.②count the number of offsprings, dead birth and live birth after parturition. Record their weight, body length, tail length, live birth and dead birth on the 1,4,7and 21 day.③calculate parental conception rate in rats, normal birth rate, and survival rate of pups at birth and breast-feeding survival.④observe fetal bone cartilage.⑤do pathological examination, if necessary, histopathological examination as well when they are sacrificed.Results:1. The transgenic tomato plant's total DNA which carrying pacA-ctxB and pacP-ctxB was amplified. After PCR and electrophoresis, approximately 450bp and 506b amplified banding appeared,corresponding to positive control group. Western Blot showed a specific band from 60KD only in the protein extraction of transgenic tomato. BCA and ELISA showed the exogenous protein of transgenic tomato carrying pacA-ctxB occupied 0.101% in the total soluble protein, and the exogenous protein of the transgenic tomato carrying pacP-ctxB occupied 0.068% in the total soluble protein.2.①Exogenous genes was found by PCR in the second generation of 2 non-transgenic tomatoes which 0.5m away from the transgenic plants;②corresponding target fragments were not detected by PCR in the total DNA of soil around 10 the transgenic tomato roots which carrying pacA-ctxB and pacP-ctxB;③the copy number of the transgenic tomato carrying pacA-ctxB is 1.3 and the pacP-ctxB is 3.2. 3.①There were no toxic symptoms from acute toxicity study and no difference between organs and positive control. The median lethal dose (LD50) of acute toxicity was larger than 20g/kg;②there were no toxic symptoms in subchronic toxicity study, and no significant difference between the test group and negative control group from hematology, biochemistry, organ index and histopathology examination.③there was no significant difference between bone marrow micronuclei incidence rates and negative control from 4 transgenic tomatoes in the bone marrow micronucleus test. However, positive control was significantly different from others(P<0.05); there was no difference between teratospermia ratio and negative control(P>0.05), but positive control was different from others(P<0.05);④there were no toxic symptoms during the experiment and no significant difference for weight(P>0.05). In generation reproductive toxicity test; there was no significance in the average weight, body length and tail length comparisons (P>0.05); there was no statistically difference in pregnancy rate, the normal delivery rate, birth survival rate and suckle survival rate compared to the control group (P>0.05); there was no statistically difference in fetuses vertebrae, sternum, ribs, bone in limbs compared to control group (P>0.05); and no anomalies were seen in the size, color, shape, hardness, and cut surface structure of heart, liver, spleen, lung, kidney, uterus, ovary, testis and epididymis observed by the naked eye.Conclusion:1. The transgenic tomato plants carrying pacA-ctxB,pacP-ctxB were obtained and the progeny of transgenic tomato plants can express exogenous proteins effectively.2. Exogenous gene pacA-ctxB,pacP-ctxB in the field could be drifted by gene flow through pollen, and the exogenous gene does not drift through the root exudates of the transgenic plants to the rhizosphere soil. The transgenic tomato plants which has high stability are low-copy transgenic plants.3. In the experiment, the transgenic tomato anti-caries vaccine has not acute toxicity,subchronic toxicity effects and it did not lead to teratogenic mutagenic response and obvious reproductive toxicity.Our experiment confirmed initially that transgenic tomato anti-caries vaccine is safe and nontoxic.