转基因番茄防龋疫苗口服免疫大白兔的实验研究
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摘要
目的：在获得含目的基因pacA-ctxB, pacP-ctxB的转基因番茄第三代种子的基础上,应用PCR、Western blot、ELISA技术对转基因番茄进行鉴定及目的蛋白浓度测定,进一步对新西兰大白兔进行口服免疫实验,初步探索表达PAcA/CTB、PAcP/CTB嵌合蛋白的转基因番茄免疫大白兔的免疫效果。
方法：本研究包括两个部分
实验一：转基因番茄中外源基因pacA-ctxB、pacP-ctxB及外源目的蛋白的检测
1.PCR法筛选含编码变异链球菌表面蛋白基因与霍乱毒素B亚单位融合基因pacA-ctxB、pacP-ctxB的转基因番茄植株。2.BCA法测定总蛋白的含量。3.Westernblot确定目的蛋白的表达。4. ELISA法检测外源目的蛋白的浓度。实验二：转基因番茄防龋疫苗口服免疫大白兔的实验研究。1.动物的选择与分组：32只新西兰大白兔随机分为A、B、C、D四大组：其中A组包含三个剂量组：A1组：以7ml/kg剂量喂食表达PAcA/CTB嵌合蛋白的转基因番茄汁(含目的蛋白53.3μg); A2组：以10ml/kg剂量喂食表达PAcA/CTB嵌合蛋白的转基因番茄汁(含目的蛋白76.20μg);A3组：以13ml/kg剂量喂食表达PAcA/CTB嵌合蛋白的转基因番茄汁(含目的蛋白99.06μg)。B组包含三个剂量组：B1组：以7ml/kg剂量喂食表达PAcP/CTB嵌合蛋白的转基因番茄汁(含目的蛋白65.62μg)；B2组：以10ml/kg剂量喂食表达PAcP/CTB嵌合蛋白的转基因番茄汁(含目的蛋白93.74μg)；B3组：以13ml/kg剂量喂食表达PAcP/CTB嵌合蛋白的转基因番茄汁(含目的蛋白121.87μg)。C组为阴性对照组：以10ml/kg剂量喂食非转基因番茄汁。D组为阳性对照组：喂食灭活全菌疫苗(1ml／次),共8个组,每组4只动物。2.免疫途径：喂食免疫。3.免疫时间：每周免疫一次,连续免疫4周。4.样品采集时间：分别于首次免疫前和免疫后第1、2、3、4、5、6、7、8、10、12周采集血液、唾液样品,测量动物体质量。5.特异性抗体的检测：血清和唾液中IgG、IgA采用ELISA法检测。6.于免疫后12周处死动物,取其心、肝、脾、肺、肾行病理学检查,观察免疫后各器官组织形态结构变化。
结果：①以转基因番茄植株总DNA为模板经PCR扩增、电泳后见约450bp或506bp、大小扩增条带,与阳性对照一致。②BCA法分别测得总蛋白含量为4.644mg/ml和3.456mg/ml,经SDS-PAGE电泳分离出分子量约60KD大小的蛋白条带,Western blot杂交证实该条带为嵌合蛋白PAcA/CTB或PAcP/CTB。目的蛋白的浓度分别为7.62μg/ml和9.37μg/ml,分别占总可溶性蛋白的0.16%和0.27%。③分别以不同剂量番茄汁喂食免疫动物后,大白兔血液和唾液中特异性抗体水平在免疫后1周逐渐升高,实验组和阳性对照组特异性抗体水平明显高于阴性对照组(P<0.05)；实验组与阳性对照组比较差异无统计学意义(P>0.05)；各剂量组间比较差异不明显；④相同时间点各组大白兔体质量比较差异无统计学意义(P>0.05)。⑤除阳性对照组心内膜出现炎细胞浸润现象外,实验组和阴性对照组未见明显的病理学改变
结论：①本课题组培育的转基因番茄成功表达了嵌合蛋白PAcA/CTB、PAcP/CTB, PAcA/CTB、PAcP/CTB含量分别占番茄果实可溶性总蛋白的0.16%和0.27%。②表达外源目的蛋白的转基因番茄免疫新西兰大白兔后,能有效的诱导动物唾液和血液中特异性抗变异链球菌表面蛋白PAcA、PAcP抗体的产生,转基因番茄表达的嵌合蛋白PAcA/CTB、PAcP/CTB具有较好的免疫原性。
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.
方法：本研究包括两个部分
实验一：转基因番茄中外源基因pacA-ctxB、pacP-ctxB及外源目的蛋白的检测
1.PCR法筛选含编码变异链球菌表面蛋白基因与霍乱毒素B亚单位融合基因pacA-ctxB、pacP-ctxB的转基因番茄植株。2.BCA法测定总蛋白的含量。3.Westernblot确定目的蛋白的表达。4. ELISA法检测外源目的蛋白的浓度。实验二：转基因番茄防龋疫苗口服免疫大白兔的实验研究。1.动物的选择与分组：32只新西兰大白兔随机分为A、B、C、D四大组：其中A组包含三个剂量组：A1组：以7ml/kg剂量喂食表达PAcA/CTB嵌合蛋白的转基因番茄汁(含目的蛋白53.3μg); A2组：以10ml/kg剂量喂食表达PAcA/CTB嵌合蛋白的转基因番茄汁(含目的蛋白76.20μg);A3组：以13ml/kg剂量喂食表达PAcA/CTB嵌合蛋白的转基因番茄汁(含目的蛋白99.06μg)。B组包含三个剂量组：B1组：以7ml/kg剂量喂食表达PAcP/CTB嵌合蛋白的转基因番茄汁(含目的蛋白65.62μg)；B2组：以10ml/kg剂量喂食表达PAcP/CTB嵌合蛋白的转基因番茄汁(含目的蛋白93.74μg)；B3组：以13ml/kg剂量喂食表达PAcP/CTB嵌合蛋白的转基因番茄汁(含目的蛋白121.87μg)。C组为阴性对照组：以10ml/kg剂量喂食非转基因番茄汁。D组为阳性对照组：喂食灭活全菌疫苗(1ml／次),共8个组,每组4只动物。2.免疫途径：喂食免疫。3.免疫时间：每周免疫一次,连续免疫4周。4.样品采集时间：分别于首次免疫前和免疫后第1、2、3、4、5、6、7、8、10、12周采集血液、唾液样品,测量动物体质量。5.特异性抗体的检测：血清和唾液中IgG、IgA采用ELISA法检测。6.于免疫后12周处死动物,取其心、肝、脾、肺、肾行病理学检查,观察免疫后各器官组织形态结构变化。
结果：①以转基因番茄植株总DNA为模板经PCR扩增、电泳后见约450bp或506bp、大小扩增条带,与阳性对照一致。②BCA法分别测得总蛋白含量为4.644mg/ml和3.456mg/ml,经SDS-PAGE电泳分离出分子量约60KD大小的蛋白条带,Western blot杂交证实该条带为嵌合蛋白PAcA/CTB或PAcP/CTB。目的蛋白的浓度分别为7.62μg/ml和9.37μg/ml,分别占总可溶性蛋白的0.16%和0.27%。③分别以不同剂量番茄汁喂食免疫动物后,大白兔血液和唾液中特异性抗体水平在免疫后1周逐渐升高,实验组和阳性对照组特异性抗体水平明显高于阴性对照组(P<0.05)；实验组与阳性对照组比较差异无统计学意义(P>0.05)；各剂量组间比较差异不明显；④相同时间点各组大白兔体质量比较差异无统计学意义(P>0.05)。⑤除阳性对照组心内膜出现炎细胞浸润现象外,实验组和阴性对照组未见明显的病理学改变
结论：①本课题组培育的转基因番茄成功表达了嵌合蛋白PAcA/CTB、PAcP/CTB, PAcA/CTB、PAcP/CTB含量分别占番茄果实可溶性总蛋白的0.16%和0.27%。②表达外源目的蛋白的转基因番茄免疫新西兰大白兔后,能有效的诱导动物唾液和血液中特异性抗变异链球菌表面蛋白PAcA、PAcP抗体的产生,转基因番茄表达的嵌合蛋白PAcA/CTB、PAcP/CTB具有较好的免疫原性。
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.
引文
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32.陈国胜.植物转基因沉默的机制及克服方法,河北农业科学,2008,12(3)：75-76.
33. Kong Q, Richter L, Yang YF, et al. Oral immunization with hepatitis B surface antigen expressed in transgenic plants[J].Proe Natl Acad Sci USA,2001,98(20):11539-11544.
34. Thanavala Y,Mahoney M,Pal S, et al.Immunogenicity in humans of an edible vaccine for hepatitis B[J],Proc Natl Acad Sci USA,2005,102(9):3378-3382.
35. Kapusta J,Modelska A,Pniewski T,et al.Oral immunization of human with transgenic lettuce expressing hepatitis surface antigen[J].Adv Exp Med Biol,2001,49(5):299-303.
36.戴敏,陈颖.口服免疫耐受的机理及应用研究进展[J].现代临床医学,2009,35(2)：85-86.
37. Smith KM, Eaton AD,Finlayson LM,et al.Oral tolerance [J].Am J Respir Crit Care Med,2000,162:175-178.
38.于善谦,王洪海,朱乃硕,等.免疫学导论[M].北京：高等教育出版社,P102-134.
39. Pearay Logra, Howard Faden,and RobertC. Vaccination Strategies for Mucosal Immune Responses [J].Clin Microbiol. Rev,2001,14:430-445.
40. Bregenholt S,Wang M.The cholera toxinB subunit is a mucosal adjuvant for oral tolerance induction in type 1 diabetes [J]. Scand J Immunol,2003,57(5):43-48.
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20.唐微.转基因拷贝数对农艺性状的影响[J].安徽农业科学,2008,36：(32),13991-13992.
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30.赵晓萌,宫飞.转基因植物中外源基因沉默机制的研究进展[J].中国农学通报,2005,21(4)：80-83
31. Jiang XL,He ZM,Peng ZQ,et al. Cholera toxin B protein in transgenic tomato fruit Induces systemic immune response in mice[J].Transgenic Res,2007,16(2):169-175.
32.陈国胜.植物转基因沉默的机制及克服方法,河北农业科学,2008,12(3)：75-76.
33. Kong Q, Richter L, Yang YF, et al. Oral immunization with hepatitis B surface antigen expressed in transgenic plants[J].Proe Natl Acad Sci USA,2001,98(20):11539-11544.
34. Thanavala Y,Mahoney M,Pal S, et al.Immunogenicity in humans of an edible vaccine for hepatitis B[J],Proc Natl Acad Sci USA,2005,102(9):3378-3382.
35. Kapusta J,Modelska A,Pniewski T,et al.Oral immunization of human with transgenic lettuce expressing hepatitis surface antigen[J].Adv Exp Med Biol,2001,49(5):299-303.
36.戴敏,陈颖.口服免疫耐受的机理及应用研究进展[J].现代临床医学,2009,35(2)：85-86.
37. Smith KM, Eaton AD,Finlayson LM,et al.Oral tolerance [J].Am J Respir Crit Care Med,2000,162:175-178.
38.于善谦,王洪海,朱乃硕,等.免疫学导论[M].北京：高等教育出版社,P102-134.
39. Pearay Logra, Howard Faden,and RobertC. Vaccination Strategies for Mucosal Immune Responses [J].Clin Microbiol. Rev,2001,14:430-445.
40. Bregenholt S,Wang M.The cholera toxinB subunit is a mucosal adjuvant for oral tolerance induction in type 1 diabetes [J]. Scand J Immunol,2003,57(5):43-48.
1. Verwoerd TC,Paridon PA,Ooyen AJ,et al.Stable accumulation of As-ergillus niger Phytase in transgenic tobacco leaves[J].J Plant Physiol,1995,109:1199-12051.
2. Simon MC,Lopez MJ.Suppressor activity of potyviral and cucumoviral infections in potyvirus-induced transgene silencing[J]. Gen Virol,2003,84(10):2877-2883.
3.任永霞.植物转化方法与概述[J].河北学院学报,2005,21(6)：38-42.
4. Ishida Y,Saito HS,et al.High efficiency transformation of maize mediated by Agrobacterium tumefaciens[J]. Nature Biotechnol,1996,14:747-750.
5. Sivamani E,Lu J,Azhakanandam K.Gene expression enhancement mediated by the 5' UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants[J]. Mol Genet Genomics.2008,279(6):563-572
6.夏兰琴,郭三堆,杀虫基因在转基因双价抗虫棉中的整合和遗传稳定性[J].科学通报,2001,(7)：46-56.
7. Haymes KM,Davis TM.Agrobacterium-mediated Transformation of Alpine and transmis to Progeny[J].Plant cell Rep,1998,17:279-283.
8. Gheysen QRaimundo V,Van Mo ntagu. Illegitimate reco mbination in plant s:Amodel for T-DNA integration[J].Gene and Devel,1991,5:287-289.
9. Zupan JR,ZambryskiP.Transfer of T-DNA forms Agrobacterium to the plant cell[J]. Plant Physiol,1995,107:1041-1047.
10.朱生伟,黄国存,孙敬三.外源DNA直接导入受体植物的研究进展[J].植物学通报,2000,17(1)：11-16.
11.魏志刚,杨传平.植物基因工程中存在的问题及对策[J].东北林业大学学报,2001,29(5)： 68-74.
12.毛万霞,许燕,张国荣,等.大豆DNA导入引起大麦籽粒蛋白含量及氨基酸含量变异的遗传稳定性分析[J].种子,2003,14(6)：6-9.
13.苏宁,吴燕民,孙丙耀,等.植物基因工程新途径：叶绿体转化[J].生物技术通报,2001(4)：9-13.
14. Liu CW, Lin CC, Jeremy J,et al.Stable chloroplast transformation in cabbage by particle bombardment[J].Plant Cell Rep,2007,26:1733-1744.
15.谷维娜,王健辉.植物转基因沉默[J].保定师范专科学校学报,2002,15(4)：20-24.
16.王关林,方宏筠等主编.植物基因工程[M].第二版,北京：科学技术出版社,2002,P46-57.
17. Elina W,Raquel L.The promoter of the Arabidopsis nuclear gene COX5b-l,encoding subunit 5b of the mitochondrial cytochromec oxidase, directs tissue-specific expression [J].Plant Molecular Biology,2004,40(5):1997-2005.
18. Ana IF,Nicolas V,Moftah A.Flexible Tools for Gene Expression and Silencing in Tomato[J].Breakthrough Technologies,2009,15(1):1729-1740.
19.贺炜华,曾富华,陈信波,等.转基因植物遗传稳定性[J].湛江师范学院学报,2008,29(6)：57-61.
20.唐微.转基因拷贝数对农艺性状的影响[J].安徽农业科学,2008,36：(32),13991-13992.
21.Bolle M,Butaye K,Goderis I,et al.The influence of matrix attachment regions on transgene expression in Arabidopsishaliana wild type and gene silencing mutants[J].Plant Molecular Biology,2006,63:533-543.
22. McCabe MS,Power JB,Lowe KC,Cocking.T-DNAtransfer,integration expression and in heritance in rice,effects of plant genotype and Agrobacterium super-virulence[J].Plant Physiol,2000,157(4):429-439.
23. Siragusa M,Carra A,Salvia L,et al.Genetic instability in calamondin plants derived from somatic embryogenesis induced by diphenylurea derivatives[J].Plant Cell Rep,2007,26: (5)1289-1296.
24. Agius F,Amaya I,Botella MA.Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression[J].Journal of Experimental Botany,2004,40(9):37-46.
25. Hileman LC,Sundstrom JF,Litt A.Molecular and Phylogenetic Analyses in Tomato[J].MADS-Box Gene Family,2006,23(11):2246-2250.
26. Stempak JM,Sohn KJ,Chiang EP,ShaneB.Cell and stage of transformation specific effects of Folate deficiency on methionine cycle.intermediates and DNA methylation in anvitromodel[J].Carcinogenesis.2005,26(5):981-990.
27. Tao Y,Wu HY,Zhang SL.Two negative cisregulatory regions involved in fruit-specific promoter activity from watermelon[J],Journal of Biologica Chemistry,2009,60(1):169-185.
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