重组抗除草剂蛋白aroA-CC-M与转Cry1Ac-M基因抗虫玉米Bt-799的食用安全性评价

发布时间：2018-05-06 00:46

本文选题：转基因食品 + 除草剂抗性　；参考：《中国疾病预防控制中心》2012年硕士论文

【摘要】：目的建立体外高效表达重组抗除草剂蛋白aroA-CC-M的方法,优化诱导表达条件,获得高纯度(95%)重组蛋白,并进行等同性分析；从毒性、致敏性两个方面对大肠杆菌原核表达系统所获得的重组抗除草剂蛋白aroA-CC-M进行食用安全性评价；采用营养成分分析、动物喂养实验等方法,观察转Cry1Ac-M基因抗虫玉米Bt-799对动物的生长发育、亚慢性毒性、营养和健康状况等的影响,对转Cry1Ac-M基因抗虫玉米Bt-799进行全面的食用安全性评价。方法 1.重组抗除草剂蛋白aroA-CC-M的体外表达、纯化和等同性分析 1.1重组抗除草剂蛋白aroA-CC-M的体外表达、纯化将克隆有aroA-CC-M基因的重组质粒pEASY-aroA-CC-M转化到大肠杆菌感受态细胞E.coli Rosetta (DE3)中,并用IPTG进行诱导表达。表达产物经过孔径为10KD的膜包浓缩后,用Ni-NTA亲和层析介质纯化,然后进行SDS-PAGE电泳检测其纯度,冻干保存。 1.2重组抗除草剂蛋白aroA-CC-M的等同性分析从分子量、N端氨基酸序列测定、免疫反应性、抗原性几个方面,将重组抗除草剂蛋白aroA-CC-M与理论蛋白进行等同性分析,分析重组抗除草剂蛋白aroA-CC-M的理论等同性。 2.重组抗除草剂蛋白aroA-CC-M的食用安全性评价 2.1毒性评价 2.1.1生物信息学分析利用生物信息学方法,将重组抗除草剂蛋白aroA-CC-M与蛋白质数据库中收录的蛋白质进行同源性分析,初步判断重组抗除草剂蛋白aroA-CC-M是否有潜在毒性。 2.1.228天喂养实验利用大肠杆菌原核表达系统表达重组抗除草剂蛋白aroA-CC-M,获得含有aroA-CC-M蛋白终浓度为0.25mg/ml、0.5mg/ml、1.0mg/ml的浓缩液,作为低、中、高剂量组,以空质粒所表达的蛋白浓缩液作为对照组。选用体重60-80g的健康断乳SD大鼠80只,雌雄各半。适应3天后,按体重随机分为4组,即对照组、2.5mg/kg bw组、5mg/kg bw组、10mg/kg bw组。蛋白浓缩液以灌胃的形式给予受试动物,以10ml/kg bw灌胃量每天灌胃1次,实验周期为28天。实验中动物单笼饲养,自由饮水、进食,观察动物的生长发育状况,每周记录1次体重和进食量。处死动物前1h对动物灌胃一次,实验结束时检测大鼠血常规、血生化、外周血淋巴细胞免疫表型分类及血液中重组抗除草剂蛋白aroA-CC-M的含量。实验结束后处死大鼠,取心、肝、脾、肾、肾上腺、胸腺、睾丸称重,计算脏器系数；对上述脏器及甲状腺、胃、十二指肠、空肠、回肠、子宫、卵巢、附睾等进行病理学检查。 2.2致敏性评价 2.2.1生物信息学分析通过全长比对、80个氨基酸片段序列相似性比对、连续8个氨基酸相同的精确比对3种方法,对重组抗除草剂蛋白aroA-CC-M的氨基酸序列与致敏原数据库中的已知致敏原进行比对,初步判断抗除草剂蛋白aroA-CC-M的潜在致敏性。 2.2.2消化稳定性分析进行重组抗除草剂蛋白aroA-CC-M在模拟胃肠液中的消化实验,分析重组抗除草剂蛋白aroA-CC-M是否具有消化抗性。 3.转Cry1Ac-M基因抗虫玉米Bt-799的食用安全性评价 3.1营养成分分析对转Cry1Ac-M基因抗虫玉米Bt-799的蛋白质、脂肪、纤维素、维生素、矿物质以及18种氨基酸进行测定,与文献中传统玉米和其他转基因玉米的主要营养成分数据进行比较,分析转Cry1Ac-M基因抗虫玉米Bt-799的主要营养成分与传统玉米以及其他转基因玉米是否有显著差异。 3.2抗营养素分析进行生物信息学分析,笔记哦转基因抗虫蛋白Cry1Ac-M与玉米中已知的抗营养因子如植酸、胰岛素酶抑制剂和胰凝乳蛋白酶抑制剂等是否有同源性,初步判断转基因抗虫蛋白Cry1Ac-M是否有抗营养作用。 3.3亚慢性毒性评价(90天喂养实验) 选用体重60-80g的健康断乳SD大鼠140只,雌雄各半。适应3天后,按体重随机分为7组,即普通饲料组、3个转基因玉米组和3个亲本玉米组。普通饲料组喂饲基础饲料,转基因玉米组分别喂饲含12.5%、25.0%和50.0%转Cry1Ac-M基因抗虫玉米Bt-799的基础料,亲本玉米组分别喂饲含12.5%、25.0%和50.0%亲本玉米郑58的基础料,连续喂养观察90天。实验中动物单笼饲养,自由饮水、进食。观察动物的生长发育状况,每周记录1次体重和进食量,实验中期检测血常规和血生化指标,实验结束时检测大鼠血常规、血生化、尿常规、凝血指标、外周血淋巴细胞免疫表型分类以及性激素水平。实验结束后处死大鼠,取心、肝、脾、肾、肾上腺、胸腺、睾丸称重,计算脏器系数；对上述脏器及胃、十二指肠、空肠、回肠、结肠、盲肠、子宫、卵巢、附睾等进行病理学检查。结果 1.重组抗除草剂蛋白aroA-CC-M的体外表达、纯化和等同性分析 1.1重组抗除草剂蛋白aroA-CC-M的体外表达、纯化利用大肠杆菌E.coli Rosetta (DE3)对重组抗除草剂蛋白aroA-CC-M进行体外表达,优化诱导表达及纯化条件,建立了采用原核表达系统获得大量可溶性重组抗除草剂蛋白aroA-CC-M的方法,获得的蛋白纯度达95%以上,产量约为15.6mg/L菌液。 1.2重组抗除草剂蛋白aroA-CC-M的等同性分析重组抗除草剂蛋白aroA-CC-M的分子量与理论值一致,N端氨基酸序列与理论序列相同,具有期望的免疫反应性和抗原性。 2.重组抗除草剂蛋白aroA-CC-M的食用安全性评价 2.1毒性评价 2.1.1生物信息学分析与Uniprot数据库中的已知蛋白质进行氨基酸序列比对,发现Uniprot数据库中与重组抗除草剂蛋白aroA-CC-M有同源性的蛋白均为抗除草剂蛋白,且没有发现发现任何一种蛋白质的信息综述中提示有毒或有抗营养作用。 2.1.228天喂养实验大鼠28天喂养实验结果表明,大鼠生长发育情况正常,转基因各剂量组与对照组在进食量、体重增长和食物利用率三个指标的组间差异均无统计学意义(P0.05)；转基因中、高剂量组个别动物的血生化、脏器系数的个别指标与对照组差异有统计学意义(P0.05),但指标的测量值均在本实验室历史性对照范围之内,且无剂量-反应关系,认为无生物学意义；血常规、外周血淋巴细胞免疫表型分类等指标差异均无统计学意义(P0.05)。 2.2致敏性评价 2.2.1生物信息学分析重组抗除草剂蛋白aroA-CC-M与已知致敏原在全长序列比对结果显示,没有发现高度序列同源性(E0.01)；80个氨基酸序列比对,没有超过35%的序列同源性的过敏原序列；连续8个氨基酸比对,没有发现连续8个氨基酸相同。说明重组抗除草剂蛋白aroA-CC-M与已知致敏原序列同源性较低。 2.2.2消化稳定性分析观察重组抗除草剂蛋白aroA-CC-M在模拟胃/肠液中的消化稳定性,结果表明重组抗除草剂蛋白aroA-CC-M在模拟胃液中易于被消化,不具有抗胃蛋白酶消化作用,但在模拟肠液中60min内不能被消化。 3.转Cry1Ac-M基因抗虫玉米Bt-799的食用安全性评价 3.1营养成分分析转Cry1Ac-M基因抗虫玉米Bt-799的蛋白质、脂肪、纤维素、维生素、矿物质和18种氨基酸等主要营养成分的含量与文献中普通玉米及其他转基因玉米的数据资料无明显差异。 3.2抗营养素分析转基因抗虫蛋白Cry1Ac-M与已知的玉米中常见的抗营养因子如植酸、胰岛素酶抑制剂和胰凝乳蛋白酶抑制剂等无同源性,初步认为转基因抗虫蛋白Cry1Ac-M无抗营养作用。 3.3亚慢性毒性评价(90天喂养实验) 大鼠90天喂养实验结果表明,大鼠生长发育情况正常,转基因玉米各剂量组与基础饲料对照组和相应的亲本对照组的进食量、体重增长及食物利用率差异无统计学意义(P0.05)；转基因玉米组的个别动物的血常规、血生化、脏器系数等个别指标与基础饲料组或相应的亲本对照组差异有统计学意义(P0.05),但指标的测量值均在本实验室历史对照范围之内,且无剂量-反应关系,认为无生物学意义；尿常规、凝血常规、外周血淋巴细胞免疫表型分类、性激素水平等方面则差异无统计学意义(P＞0.05)。病理学检查未见异常。结论 1.对重组抗除草剂蛋白aroA-CC-M的体外表达和纯化进行了研究,通过对诱导表达条件和纯化方法的摸索、优化,建立了采用原核表达系统获得高纯度(95%)可溶性重组抗除草剂蛋白aroA-CC-M的有效方法,产量为15.6mg/L菌液。该方法操作简单、易于扩大生产,为进一步对外源蛋白的评价奠定了基础。体外表达所得的重组抗除草剂蛋白aroA-CC-M与理论蛋白具有等同性,可用于食用安全性评价研究中。 2.生物信息学研究证实,重组抗除草剂蛋白aroA-CC-M与已知毒蛋白、抗营养素或致敏原不具有同源性；首次对大肠杆菌体外表达系统获得的重组抗除草剂蛋白aroA-CC-M进行了大鼠28天喂养实验,结果显示转基因组的各项指标与基础饲料对照组和亲本对照组相比,差异无统计学意义,未发现对大鼠有毒性作用；重组抗除草剂蛋白aroA-CC-M在模拟胃液中易于被消化,不具有抗胃蛋白酶消化作用,但在模拟肠液中60min内不能被消化。 3.转Cry1Ac-M基因抗虫玉米Bt-799的主要营养素含量与文献中报道的传统玉米和其他转基因玉米的数值无统计学差异；转基因抗虫蛋白Cry1Ac-M与已知抗营养因子无同源性,认为无抗营养作用,说明Cry1Ac-M基因的转入对玉米的营养价值无明显影响；大鼠亚慢性毒性实验结果未显示转Cry1Ac-M基因抗虫玉米Bt-799对大鼠有毒性作用。
[Abstract]:Purpose

establishing a method for efficiently expressing recombinant anti - herbicide protein aroA - CC - M in vitro , optimizing induction expression condition , obtaining high - purity ( 95 % ) recombinant protein , and carrying out equivalence analysis ;
the recombinant anti - herbicide protein aroA - CC - M obtained by the E . coli prokaryotic expression system is evaluated for edible safety from two aspects of toxicity and sensitization ;
The effects of Cry1Ac - M gene resistant maize Bt - 799 on growth and development , subchronic toxicity , nutrition and health status were observed by means of nutritional composition analysis and animal feeding experiments .

method

1 . In vitro expression , purification and equivalence analysis of recombinant anti - herbicide protein aroA - CC - M

1.1 In vitro expression and purification of recombinant anti - herbicide protein aroA - CC - M

The recombinant plasmid of aroA - CC - M gene was transformed into E . coli Rosetta ( DE3 ) .

1.2 Isomorphism analysis of recombinant anti - herbicide protein aroA - CC - M

The equivalence of the recombinant anti - herbicide protein aroA - CC - M with the theoretical protein was analyzed from the aspects of molecular weight , N - terminal amino acid sequence determination , immunoreactivity and antigenicity , and the theoretical equivalence of the recombinant anti - herbicide protein aroA - CC - M was analyzed .

2 . Safety evaluation of recombinant anti - herbicide protein aroA - CC - M

2.1 Toxicity evaluation

2.1 . 1 Bioinformatic Analysis

The recombinant anti - herbicide protein aroA - CC - M was analyzed by bioinformatics to determine whether the recombinant anti - herbicide protein aroA - CC - M had potential toxicity .

2.1 . 228 Day feeding experiment

The recombinant anti - herbicide protein aroA - CC - M was expressed by E . coli prokaryotic expression system . The final concentration of protein concentrate containing aroA - CC - M was 0.25 mg / ml , 0.5 mg / ml and 1.0 mg / ml . The rats were randomly divided into 4 groups : control group , 2.5 mg / kg bw group , 5 mg / kg bw group and 10 mg / kg bw group .
The above organs and thyroid , stomach , duodenum , jejunum , ileum , uterus , ovary , appendix , etc . are examined by pathology .

2.2 Sensitization Evaluation

2.2 . 1 Bioinformatic Analysis

The amino acid sequence of the recombinant anti - herbicide protein aroA - CC - M was compared with the known sensitizer in the allergen database through the comparison of the amino acid sequence of the 80 amino acid fragment sequences and the amino acid sequence of the 80 amino acid fragment sequences by the full - length ratio pair , and the potential sensitization of the anti - herbicide protein aroA - CC - M was preliminarily determined .

2.2 . 2 Analysis of digestion stability

The digestion experiments of recombinant anti - herbicide protein aroA - CC - M in simulated gastrointestinal fluids were carried out to determine whether the recombinant anti - herbicide protein aroA - CC - M had digestive resistance .

3 . Food Safety Evaluation of Transgenic Bt - 799 Transgenic Cry1Ac - M Gene

3.1 Analysis of nutrient components

The protein , fat , cellulose , vitamins , minerals and 18 amino acids of Cry1Ac - M gene resistant maize Bt - 799 were measured . The main nutrient components of Cry1Ac - M gene resistant maize Bt - 799 were compared with those of traditional corn and other transgenic maize .

3.2 Anti - nutrient analysis

The bioinformatic analysis , notes , transgenic anti - insect protein Cry1Ac - M and the known anti - nutritional factors such as phytic acid , insulin enzyme inhibitor and trypsin inhibitor in corn have homology with each other , and whether the transgenic anti - insect protein Cry1Ac - M has anti - nutrition effect is preliminarily judged .

3.3 Subchronic Toxicity Assessment ( 90 Day Feeding Experiment )

The rats were fed with diets containing 12.5 % , 25.0 % and 50.0 % of Cry1Ac - M gene .
pathological examination is carried out on the viscera and stomach , duodenum , jejunum , ileum , colon , cecum , uterus , ovary , appendix , and the like .

Results

1 . In vitro expression , purification and equivalence analysis of recombinant anti - herbicide protein aroA - CC - M

1.1 In vitro expression and purification of recombinant anti - herbicide protein aroA - CC - M

The recombinant anti - herbicide protein aroA - CC - M was expressed in vitro by E . coli Rosetta ( DE3 ) , and the expression and purification conditions were optimized . A large amount of soluble recombinant anti - herbicide protein aroA - CC - M was obtained by prokaryotic expression system . The purity of the protein obtained was over 95 % and the yield was about 15.6 mg / L .

1.2 Isomorphism analysis of recombinant anti - herbicide protein aroA - CC - M

The molecular weight of the recombinant anti - herbicide protein aroA - CC - M is consistent with the theoretical value , and the N - terminal amino acid sequence is the same as the theoretical sequence , and has the desired immunoreactivity and antigenicity .

2 . Safety evaluation of recombinant anti - herbicide protein aroA - CC - M

2.1 Toxicity evaluation

2.1 . 1 Bioinformatic Analysis

In comparison with known proteins in the Uniproot database , it was found that the proteins homologous to the recombinant anti - herbicide protein aroA - CC - M in the Univ database were anti - herbicide proteins , and no toxic or anti - nutritional effects were noted in the information review of any protein found .

2.1 . 228 Day feeding experiment

The results of 28 - day feeding experiment in rats showed that the growth and development of rats were normal , and there was no significant difference between the three indexes ( P0.05 ) .
In the transgenic , the individual indexes of blood biochemistry and organ coefficients of the high - dose group were significantly different from those in the control group ( P0.05 ) , but the measured values of the indexes were within the historical control range of the laboratory , and there was no dose - response relationship , which was considered to be of no biological significance ;
There was no significant difference between the blood routine and the immune phenotype of peripheral blood lymphocytes ( P0.05 ) .

2.2 Sensitization Evaluation

2.2 . 1 Bioinformatic Analysis

The results showed that the recombinant anti - herbicide protein aroA - CC - M did not show a high degree of homology ( E0 . 01 ) .
80 amino acid sequences with no more than 35 % sequence homology ;
Eight consecutive amino acid sequences were found to be identical to each other , and the homology of the recombinant anti - herbicide protein aroA - CC - M with the known sensitizing original sequence was low .

2.2 . 2 Analysis of digestion stability

The digestion stability of recombinant anti - herbicide protein aroA - CC - M in simulated gastric juice was observed . The results showed that the recombinant anti - herbicide protein aroA - CC - M was easy to digest in the simulated gastric juice without anti - pepsin digestion , but could not be digested in the simulated intestinal fluid for 60 minutes .

3 . Food Safety Evaluation of Transgenic Bt - 799 Transgenic Cry1Ac - M Gene

3.1 Analysis of nutrient components

The contents of protein , fat , cellulose , vitamins , minerals and 18 amino acids of Cry1Ac - M gene resistant maize Bt - 799 were not significantly different from those of common maize and other transgenic maize in the literature .

3.2 Anti - nutrient analysis

The transgenic insect - resistant protein Cry1Ac - M has no homology with common anti - nutritional factors such as phytic acid , insulin enzyme inhibitor and trypsin inhibitor in the known corn , and the transgenic insect - resistant protein Cry1Ac - M has no anti - nutrition effect .

3.3 Subchronic Toxicity Assessment ( 90 Day Feeding Experiment )

The results of 90 - day feeding experiment in rats showed that the growth and development of rats were normal , and there was no significant difference between the feeding quantity , body weight gain and food utilization ratio of the transgenic corn and the basal diet control group and the corresponding parent control group ( P0.05 ) .
The blood routine , blood biochemistry , organ coefficient and other individual indexes of the transgenic corn group were statistically significant ( P0.05 ) , but the measured values of the indexes were within the historical control range of the laboratory , and there was no dose - response relationship , which was considered to be of no biological significance ;
There was no significant difference in urine routine , blood coagulation routine , peripheral blood lymphocyte immune phenotype classification and sex hormone level ( P > 0.05 ) . The pathological examination was not abnormal .

Conclusion

1 . In vitro expression and purification of recombinant anti - herbicide protein aroA - CC - M were studied . The effective method for obtaining high purity ( 95 % ) soluble recombinant anti - herbicide protein aroA - CC - M was established by using prokaryotic expression system . The yield was 15.6 mg / L . The method is simple and easy to expand and produce . The recombinant anti - herbicide protein aroA - CC - M expressed in vitro has the same equivalence with the theoretical protein , which can be used in the study of edible safety evaluation .

2 . Bioinformatic studies confirm that the recombinant anti - herbicide protein aroA - CC - M has no homology with known toxin proteins , anti - nutrients or allergens ;
For the first time , the recombinant anti - herbicide protein aroA - CC - M obtained by the in vitro expression system of E . coli was fed to the rats for 28 days , and the results showed that the indexes of the transgenic genome were not statistically significant compared with the basal diet control group and the parent control group , and no toxic effect on the rats was found ;
The recombinant anti - herbicide protein aroA - CC - M is easy to digest in the simulated gastric juice , does not have anti - pepsin digestion , but cannot be digested in the simulated intestinal fluid for 60 minutes .

3 . The main nutrient content of Cry1Ac - M gene resistant maize Bt - 799 was not significantly different from those reported in the literature .
The transgenic insect - resistant protein Cry1Ac - M has no homology with known anti - nutritional factors , and is considered to have no anti - nutrition effect , and indicates that the transformation of Cry1Ac - M gene has no obvious effect on the nutritional value of corn ;
The experimental results of subchronic toxicity in rats did not show that the transgenic Bt - 799 transgenic Cry1Ac - M gene had a toxic effect on rats .

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155

【参考文献】