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The Prelliminary Study of the Safety on the Transgenic Tomato Anti-caries Vaccine

Author: BaiGuoHui
Tutor: LiuJianGuo；YangDeQin；GuZuo
School: Zunyi Medical College,
Course: Stomatology
Keywords: Transgenic tomato Genetic drift Security Anti-caries vaccine Streptococcus mutans Caries
CLC: R781.1
Type: Master's thesis
Year: 2010
Downloads: 95
Quote: 2
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Abstract

Objective: To observe the exogenous purpose of gene pacA-ctxB pacP-ctxB and purpose protein PAcA / CTB PAcP / CTB in transgenic tomato and non-transgenic gene plant expression of transferred genes tomato immune animals after the toxic role, preliminary analysis of transgenic tomato anti- The caries vaccine ecological security and safety of the animals. Methods: In this study, including the three-part experiment a turn the transgenic tomato exogenous purposes genes and the purpose of protein expression detected extract turn transgenic tomato total of DNA PCR screening containing exogenous gene pacA-ctxB pacP-ctxB the transgenic tomato plants; detected by Western blot expression of the exogenous protein; determination of the total protein content of tomato fruit, extract of tomato fruit protein with BCA kit and foreign source target protein content was measured by ELISA. Experiment II turn transgenic tomato's ecological security check, exogenous genes with pollen drift of detection: field planting transferred genes tomato, from the turn of transgenic plants the edge of 0.5m, 1m, 3m, 10m at planting non-transfected gene control tomato gene flow receptor , flowering freely pollination, the results at a distance of transgenic tomato plants edge 0.5m, 1m, 3m and 10m, were randomized to receive 10 tomato fruit, collecting seed laboratory-grown seedlings of collecting leaves for PCR detection. Turn detection of transgenic plants rhizosphere soil: each take a soil sample in transgenic tomato plant roots, east, south, west, north, and total DNA extracted soil the exogenous gene roots drift detection. 3, turn genes tomato purpose of gene copy number of detection: our group pre-construct the recombinant plasmid pEAC10, pEPC10 as standard of SYBR Green real-time fluorescence quantitative PCR method with exogenous gene pacA-ctxB, pacP-ctxB of transgenic tomato plants a sample of the total genomic repeat testing, the average as the purpose of gene copy number. Experiments transgenic tomato animal safety check, Acute toxicity test on rats (1) Animal selection, grouping: wistar rats 24, male and female, were randomly divided into three groups. Group A: of transgenic tomato juice containing PAcA / CTB protein immunization; B group: use transgenic tomato juice containing PAcP / CTB protein immunohistochemistry; C group (control group): immunization with non-transgenic tomato juice. (2) immunization program: three times a day, at 20g/kg (weight) dose gavage. (3) OUTCOME MEASURES: (1) every two days during the experiment measuring animal weight. ② records toxicity symptoms and disappearance of time, the time of death. ③ All animals were sacrificed on the 7th day, heart, liver, kidney, spleen, thymus, histopathological examination. 2, rat subchronic toxicity test (1) selection of animals, grouping: 72 of wistar rats were randomly divided into six groups. Experimental group A1: transgenic tomato juice containing PAcA / CTB protein immunization dose of 20g/kg (weight); experimental group A2: dose immunization with the transgenic tomato juice containing PAcA / CTB protein to 10g/kg (Weight); experimental group B1: with the transgenic tomato juice containing PAcP / CTB protein 20g/kg (weight) dose immunization; experimental group B2: with the transgenic tomato juice containing PAcP / CTB protein immunization dose of 10g/kg (weight); experimental group C1: regular daily dose immunization with non-transgenic tomato juice 20g/kg (weight); experimental group C2: 10g/kg (weight) dose immunization with non-transgenic tomato juice; (2) immunization program: 30 days intragastrically gavage. (3) OUTCOME MEASURES: ① experiments during the measurement of animal body weight per day. (2) close observation of the activities of animal behavior and the appearance of signs. The ③ gavage 30 days to hematology, blood chemistry, and urine tests. ④ whichever is the heart, liver, spleen, lung, kidney weighed and calculated organ coefficient. ⑤ heart, liver, spleen, lung, kidney histopathological examination. 3, check mutagenesis experiments 3.1 bone marrow micronucleus assay: (1) Animal selection, grouping 42: Kunming mice were randomly divided into 7 groups. Experimental group A1: the transgenic tomato juice containing PAcA / CTB protein 5g/kg (weight) dose immune; experimental group A2: dose immunization with transgenic tomato juice containing PAcA / CTB protein 2g/kg (weight); experimental group B1: with the transgenic tomato juice containing PAcP / CTB protein 5g/kg (body weight) dose immunization; experimental group B2: with the transgenic tomato juice containing PAcP / CTB protein dose immune to 2g/kg (weight); negative control group C1 : non-transgenic tomato juice 5g/kg (weight) dose immunization; negative control group C2: 2g/kg (body weight) dose immunization with non-transgenic tomato juice; positive control group D: with 100mg / (kg * d ) injection of cyclophosphamide; (2) immunization program: the experimental group and the control group intragastrically four days, the positive control cyclophosphamide intraperitoneal injection, four days in a row. Five days the animals were sacrificed. (3) OUTCOME MEASURES: Preparation of mouse bone marrow films, under a microscope counting micronucleated addicted to the number of polychromatic erythrocytes averaged. Calculate the rate of micronuclei. 3.2 sperm deformity experiments: (1) Animal selection, grouping: 42 male mice were randomly divided into 7 groups with cyclophosphamide injections of 20mg / (kg * d) In addition to the positive control group D, the other grouping with bone marrow cell micronucleus experimental. (2) immunization program: the experimental group and the control group intragastrically five days, the positive control group, cyclophosphamide by intraperitoneal injection for 5 consecutive days. The animals were sacrificed 35 days after the first administration of the test substance. (3) OUTCOME MEASURES: Preparation of mouse sperm piece count malformed sperm under the microscope, calculation of sperm deformity rate. The 4 generation reproductive toxicity test (1) selection of animals, grouping 60: SD rats were randomly divided into three groups. Group A: of transgenic tomato juice containing PAcA / CTB protein immunization; B group: use transgenic tomato juice containing PAcP / CTB protein immunization; C group (control group): immunization with non-transgenic tomato juice. (2) immunization program: 1g/kg (body weight) dose, males continued After 8 weeks, the females start gavage for 2 weeks, the male and female rats 1:1 mated, mating continued during irrigation stomach, male killed, three weeks later, females continue orally until three weeks after the birth of the offspring weaning. (3) OUTCOME MEASURES: (1) observed the general condition of the rats daily, weekly weighing. (2) each pregnancy cubs number of animals examined after delivery, dead litter size, the number of pigs born alive. Record the pups were born and 1,4,7,21 days after birth, weight, body length and tail length, and count live, the number of dead pups. ③ calculate the parental rat pregnancy rate, normal delivery rate, and the pups survival at birth and lactation survival rate. The ④ observed fetal rat bone cartilage. ⑤ all animals were killed naked eye pathological examination, histopathological examination is necessary. Results: 1, and the total gene of the transgenic tomato containing pacA-CtxB and pacP-ctxB conduct PCR amplification to obtain a size of approximately 450bp and 506 bp of the strip, is consistent with the positive control. Western Blot analysis of transgenic tomato protein extracted sample about 60KD PVDF membrane at specific bands. BCA and ELISA method measured containing PAcA / CTB transfer genes tomato exogenous target protein accounting for 0.101% of the total protein content of the plant containing PAcP / CTB transferred gene in tomato exogenous protein accounting for 0.068% of the total protein content of the plant. PCR detection ① distance turn genetic tomato 0.5m at the 17 non-transgenic tomato second-generation plants have exogenous purpose of gene pacA-ctxB, pacP-ctxB; ② The respectively 10 containing pacA-ctxB, pacP-ctxB turn genes four directions of east, south, west and north of the tomato rhizosphere soil total DNA not amplified target fragment; ③ The research group nurtured pacA-ctxB transgenic tomato plants exogenous gene copy number was 1.3, containing transgenic tomato plants pacP-ctxB exogenous gene copy number was 3.2 ① acute toxicity experiments in rats showed no toxic symptoms, the experimental rats organs with the negative control group differences were not statistically significant, acute LD50 in toxic median lethal dose gt; 20g/kg; ② The sub-chronic toxicity test in male and female rats showed no toxic symptoms, hematology and biochemistry, organ coefficient, and histopathologic examination, experimental rats with the negative control group was no significant difference (P gt; 0.05); ③ The bone marrow micronucleus experiment 4 turn gene the tomato experimental group mice bone marrow cells micronucleus incidence of negative control group, the difference was not statistically significance (P gt; 0.05) difference: the positive control group and the other groups were statistically significant (P lt; 0.05); sperm deformity test 4 turn genes tomato experimental group tested mice sperm abnormality rate of the negative control group, the difference was not statistically significant (P gt; 0.05); while the positive control group differences were statistically significant (P lt; 0.05) compared with other groups; the ④ generation reproductive toxicity experiments, the rats during the experiment were not toxic symptoms the average weight of the pups, length, tail length, weight change difference was not statistically significant (P gt; 0.05); conception of the experimental group by pairwise comparisons showed no significant difference (P gt; 0.05); rate, normal delivery rate, the pups were born survival, the survival rate of cubs breastfeeding indicators with the control group, the difference was not statistically significant (P gt; 0.05); experimental group fetal spine, sternum, ribs, limbs and other tests and the control group, the difference was not statistically significant (P gt; 0.05); observed by the naked eye, each group of animal heart, liver, spleen, lung, kidney, uterus, ovary, testis, epididymis size, color, shape, softness and section structure, etc. showed no abnormal changes. Conclusion: 1, to obtain transgenic tomato plants containing pacA-ctxB pacP-ctxB target gene, and progeny plants of transgenic tomato exogenous protein expression. 2, the exogenous gene pacA-ctxB pacP-ctxB in the field can be caused by pollen gene flow, but exogenous gene not root exudates drift, the transgenic plants rhizosphere soil. Transgenic tomato plants built by our group is a low-copy transgenic plants, has good stability. 3, the experimental transgenic tomato anticaries vaccine no acute, subchronic toxicity, it will not cause teratogenic mutations reaction and obvious reproductive toxicity, initially confirmed that the transgenic tomato anti-caries vaccine is safe and non-toxic.

The Prelliminary Study of the Safety on the Transgenic Tomato Anti-caries Vaccine

Abstract

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