本文選題：轉(zhuǎn)基因食品 + 除草劑抗性�。� 參考：《中國疾病預(yù)防控制中心》2012年碩士論文

【摘要】：目的 建立體外高效表達(dá)重組抗除草劑蛋白aroA-CC-M的方法,優(yōu)化誘導(dǎo)表達(dá)條件,獲得高純度(95%)重組蛋白,并進(jìn)行等同性分析；從毒性、致敏性兩個方面對大腸桿菌原核表達(dá)系統(tǒng)所獲得的重組抗除草劑蛋白aroA-CC-M進(jìn)行食用安全性評價；采用營養(yǎng)成分分析、動物喂養(yǎng)實驗等方法,觀察轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799對動物的生長發(fā)育、亞慢性毒性、營養(yǎng)和健康狀況等的影響,對轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799進(jìn)行全面的食用安全性評價。 方法 1.重組抗除草劑蛋白aroA-CC-M的體外表達(dá)、純化和等同性分析 1.1重組抗除草劑蛋白aroA-CC-M的體外表達(dá)、純化 將克隆有aroA-CC-M基因的重組質(zhì)粒pEASY-aroA-CC-M轉(zhuǎn)化到大腸桿菌感受態(tài)細(xì)胞E.coli Rosetta (DE3)中,并用IPTG進(jìn)行誘導(dǎo)表達(dá)。表達(dá)產(chǎn)物經(jīng)過孔徑為10KD的膜包濃縮后,用Ni-NTA親和層析介質(zhì)純化,然后進(jìn)行SDS-PAGE電泳檢測其純度,凍干保存。 1.2重組抗除草劑蛋白aroA-CC-M的等同性分析 從分子量、N端氨基酸序列測定、免疫反應(yīng)性、抗原性幾個方面,將重組抗除草劑蛋白aroA-CC-M與理論蛋白進(jìn)行等同性分析,分析重組抗除草劑蛋白aroA-CC-M的理論等同性。 2.重組抗除草劑蛋白aroA-CC-M的食用安全性評價 2.1毒性評價 2.1.1生物信息學(xué)分析 利用生物信息學(xué)方法,將重組抗除草劑蛋白aroA-CC-M與蛋白質(zhì)數(shù)據(jù)庫中收錄的蛋白質(zhì)進(jìn)行同源性分析,初步判斷重組抗除草劑蛋白aroA-CC-M是否有潛在毒性。 2.1.228天喂養(yǎng)實驗 利用大腸桿菌原核表達(dá)系統(tǒng)表達(dá)重組抗除草劑蛋白aroA-CC-M,獲得含有aroA-CC-M蛋白終濃度為0.25mg/ml、0.5mg/ml、1.0mg/ml的濃縮液,作為低、中、高劑量組,以空質(zhì)粒所表達(dá)的蛋白濃縮液作為對照組。選用體重60-80g的健康斷乳SD大鼠80只,雌雄各半。適應(yīng)3天后,按體重隨機分為4組,即對照組、2.5mg/kg bw組、5mg/kg bw組、10mg/kg bw組。蛋白濃縮液以灌胃的形式給予受試動物,以10ml/kg bw灌胃量每天灌胃1次,實驗周期為28天。實驗中動物單籠飼養(yǎng),自由飲水、進(jìn)食,觀察動物的生長發(fā)育狀況,每周記錄1次體重和進(jìn)食量。處死動物前1h對動物灌胃一次,實驗結(jié)束時檢測大鼠血常規(guī)、血生化、外周血淋巴細(xì)胞免疫表型分類及血液中重組抗除草劑蛋白aroA-CC-M的含量。實驗結(jié)束后處死大鼠,取心、肝、脾、腎、腎上腺、胸腺、睪丸稱重,計算臟器系數(shù)；對上述臟器及甲狀腺、胃、十二指腸、空腸、回腸、子宮、卵巢、附睪等進(jìn)行病理學(xué)檢查。 2.2致敏性評價 2.2.1生物信息學(xué)分析 通過全長比對、80個氨基酸片段序列相似性比對、連續(xù)8個氨基酸相同的精確比對3種方法,對重組抗除草劑蛋白aroA-CC-M的氨基酸序列與致敏原數(shù)據(jù)庫中的已知致敏原進(jìn)行比對,初步判斷抗除草劑蛋白aroA-CC-M的潛在致敏性。 2.2.2消化穩(wěn)定性分析 進(jìn)行重組抗除草劑蛋白aroA-CC-M在模擬胃腸液中的消化實驗,分析重組抗除草劑蛋白aroA-CC-M是否具有消化抗性。 3.轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799的食用安全性評價 3.1營養(yǎng)成分分析 對轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799的蛋白質(zhì)、脂肪、纖維素、維生素、礦物質(zhì)以及18種氨基酸進(jìn)行測定,與文獻(xiàn)中傳統(tǒng)玉米和其他轉(zhuǎn)基因玉米的主要營養(yǎng)成分?jǐn)?shù)據(jù)進(jìn)行比較,分析轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799的主要營養(yǎng)成分與傳統(tǒng)玉米以及其他轉(zhuǎn)基因玉米是否有顯著差異。 3.2抗?fàn)I養(yǎng)素分析 進(jìn)行生物信息學(xué)分析,筆記哦轉(zhuǎn)基因抗蟲蛋白Cry1Ac-M與玉米中已知的抗?fàn)I養(yǎng)因子如植酸、胰島素酶抑制劑和胰凝乳蛋白酶抑制劑等是否有同源性,初步判斷轉(zhuǎn)基因抗蟲蛋白Cry1Ac-M是否有抗?fàn)I養(yǎng)作用。 3.3亞慢性毒性評價(90天喂養(yǎng)實驗) 選用體重60-80g的健康斷乳SD大鼠140只,雌雄各半。適應(yīng)3天后,按體重隨機分為7組,即普通飼料組、3個轉(zhuǎn)基因玉米組和3個親本玉米組。普通飼料組喂飼基礎(chǔ)飼料,轉(zhuǎn)基因玉米組分別喂飼含12.5%、25.0%和50.0%轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799的基礎(chǔ)料,親本玉米組分別喂飼含12.5%、25.0%和50.0%親本玉米鄭58的基礎(chǔ)料,連續(xù)喂養(yǎng)觀察90天。實驗中動物單籠飼養(yǎng),自由飲水、進(jìn)食。觀察動物的生長發(fā)育狀況,每周記錄1次體重和進(jìn)食量,實驗中期檢測血常規(guī)和血生化指標(biāo),實驗結(jié)束時檢測大鼠血常規(guī)、血生化、尿常規(guī)、凝血指標(biāo)、外周血淋巴細(xì)胞免疫表型分類以及性激素水平。實驗結(jié)束后處死大鼠,取心、肝、脾、腎、腎上腺、胸腺、睪丸稱重,計算臟器系數(shù)；對上述臟器及胃、十二指腸、空腸、回腸、結(jié)腸、盲腸、子宮、卵巢、附睪等進(jìn)行病理學(xué)檢查。 結(jié)果 1.重組抗除草劑蛋白aroA-CC-M的體外表達(dá)、純化和等同性分析 1.1重組抗除草劑蛋白aroA-CC-M的體外表達(dá)、純化 利用大腸桿菌E.coli Rosetta (DE3)對重組抗除草劑蛋白aroA-CC-M進(jìn)行體外表達(dá),優(yōu)化誘導(dǎo)表達(dá)及純化條件,建立了采用原核表達(dá)系統(tǒng)獲得大量可溶性重組抗除草劑蛋白aroA-CC-M的方法,獲得的蛋白純度達(dá)95%以上,產(chǎn)量約為15.6mg/L菌液。 1.2重組抗除草劑蛋白aroA-CC-M的等同性分析 重組抗除草劑蛋白aroA-CC-M的分子量與理論值一致,N端氨基酸序列與理論序列相同,具有期望的免疫反應(yīng)性和抗原性。 2.重組抗除草劑蛋白aroA-CC-M的食用安全性評價 2.1毒性評價 2.1.1生物信息學(xué)分析 與Uniprot數(shù)據(jù)庫中的已知蛋白質(zhì)進(jìn)行氨基酸序列比對,發(fā)現(xiàn)Uniprot數(shù)據(jù)庫中與重組抗除草劑蛋白aroA-CC-M有同源性的蛋白均為抗除草劑蛋白,且沒有發(fā)現(xiàn)發(fā)現(xiàn)任何一種蛋白質(zhì)的信息綜述中提示有毒或有抗?fàn)I養(yǎng)作用。 2.1.228天喂養(yǎng)實驗 大鼠28天喂養(yǎng)實驗結(jié)果表明,大鼠生長發(fā)育情況正常,轉(zhuǎn)基因各劑量組與對照組在進(jìn)食量、體重增長和食物利用率三個指標(biāo)的組間差異均無統(tǒng)計學(xué)意義(P0.05)；轉(zhuǎn)基因中、高劑量組個別動物的血生化、臟器系數(shù)的個別指標(biāo)與對照組差異有統(tǒng)計學(xué)意義(P0.05),但指標(biāo)的測量值均在本實驗室歷史性對照范圍之內(nèi),且無劑量-反應(yīng)關(guān)系,認(rèn)為無生物學(xué)意義；血常規(guī)、外周血淋巴細(xì)胞免疫表型分類等指標(biāo)差異均無統(tǒng)計學(xué)意義(P0.05)。 2.2致敏性評價 2.2.1生物信息學(xué)分析 重組抗除草劑蛋白aroA-CC-M與已知致敏原在全長序列比對結(jié)果顯示,沒有發(fā)現(xiàn)高度序列同源性(E0.01)；80個氨基酸序列比對,沒有超過35%的序列同源性的過敏原序列；連續(xù)8個氨基酸比對,沒有發(fā)現(xiàn)連續(xù)8個氨基酸相同。說明重組抗除草劑蛋白aroA-CC-M與已知致敏原序列同源性較低。 2.2.2消化穩(wěn)定性分析 觀察重組抗除草劑蛋白aroA-CC-M在模擬胃/腸液中的消化穩(wěn)定性,結(jié)果表明重組抗除草劑蛋白aroA-CC-M在模擬胃液中易于被消化,不具有抗胃蛋白酶消化作用,但在模擬腸液中60min內(nèi)不能被消化。 3.轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799的食用安全性評價 3.1營養(yǎng)成分分析 轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799的蛋白質(zhì)、脂肪、纖維素、維生素、礦物質(zhì)和18種氨基酸等主要營養(yǎng)成分的含量與文獻(xiàn)中普通玉米及其他轉(zhuǎn)基因玉米的數(shù)據(jù)資料無明顯差異。 3.2抗?fàn)I養(yǎng)素分析 轉(zhuǎn)基因抗蟲蛋白Cry1Ac-M與已知的玉米中常見的抗?fàn)I養(yǎng)因子如植酸、胰島素酶抑制劑和胰凝乳蛋白酶抑制劑等無同源性,初步認(rèn)為轉(zhuǎn)基因抗蟲蛋白Cry1Ac-M無抗?fàn)I養(yǎng)作用。 3.3亞慢性毒性評價(90天喂養(yǎng)實驗) 大鼠90天喂養(yǎng)實驗結(jié)果表明,大鼠生長發(fā)育情況正常,轉(zhuǎn)基因玉米各劑量組與基礎(chǔ)飼料對照組和相應(yīng)的親本對照組的進(jìn)食量、體重增長及食物利用率差異無統(tǒng)計學(xué)意義(P0.05)；轉(zhuǎn)基因玉米組的個別動物的血常規(guī)、血生化、臟器系數(shù)等個別指標(biāo)與基礎(chǔ)飼料組或相應(yīng)的親本對照組差異有統(tǒng)計學(xué)意義(P0.05),但指標(biāo)的測量值均在本實驗室歷史對照范圍之內(nèi),且無劑量-反應(yīng)關(guān)系,認(rèn)為無生物學(xué)意義；尿常規(guī)、凝血常規(guī)、外周血淋巴細(xì)胞免疫表型分類、性激素水平等方面則差異無統(tǒng)計學(xué)意義(P＞0.05)。病理學(xué)檢查未見異常。 結(jié)論 1.對重組抗除草劑蛋白aroA-CC-M的體外表達(dá)和純化進(jìn)行了研究,通過對誘導(dǎo)表達(dá)條件和純化方法的摸索、優(yōu)化,建立了采用原核表達(dá)系統(tǒng)獲得高純度(95%)可溶性重組抗除草劑蛋白aroA-CC-M的有效方法,產(chǎn)量為15.6mg/L菌液。該方法操作簡單、易于擴大生產(chǎn),為進(jìn)一步對外源蛋白的評價奠定了基礎(chǔ)。體外表達(dá)所得的重組抗除草劑蛋白aroA-CC-M與理論蛋白具有等同性,可用于食用安全性評價研究中。 2.生物信息學(xué)研究證實,重組抗除草劑蛋白aroA-CC-M與已知毒蛋白、抗?fàn)I養(yǎng)素或致敏原不具有同源性；首次對大腸桿菌體外表達(dá)系統(tǒng)獲得的重組抗除草劑蛋白aroA-CC-M進(jìn)行了大鼠28天喂養(yǎng)實驗,結(jié)果顯示轉(zhuǎn)基因組的各項指標(biāo)與基礎(chǔ)飼料對照組和親本對照組相比,差異無統(tǒng)計學(xué)意義,未發(fā)現(xiàn)對大鼠有毒性作用；重組抗除草劑蛋白aroA-CC-M在模擬胃液中易于被消化,不具有抗胃蛋白酶消化作用,但在模擬腸液中60min內(nèi)不能被消化。 3.轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799的主要營養(yǎng)素含量與文獻(xiàn)中報道的傳統(tǒng)玉米和其他轉(zhuǎn)基因玉米的數(shù)值無統(tǒng)計學(xué)差異；轉(zhuǎn)基因抗蟲蛋白Cry1Ac-M與已知抗?fàn)I養(yǎng)因子無同源性,認(rèn)為無抗?fàn)I養(yǎng)作用,說明Cry1Ac-M基因的轉(zhuǎn)入對玉米的營養(yǎng)價值無明顯影響；大鼠亞慢性毒性實驗結(jié)果未顯示轉(zhuǎn)Cry1Ac-M基因抗蟲玉米Bt-799對大鼠有毒性作用。
[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 .

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R155

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王進(jìn)忠,孫淑玲,楊寶東;轉(zhuǎn)Bt基因植物的研究與應(yīng)用前景[J];北京農(nóng)學(xué)院學(xué)報;2002年03期

2 于旭華,馮定遠(yuǎn),王芳;酶制劑在玉米—豆粕型日糧中的應(yīng)用[J];飼料工業(yè);2002年12期

3 易學(xué)武,張石蕊;糙米與玉米的營養(yǎng)成分比較及其在畜禽日糧中的應(yīng)用[J];飼料工業(yè);2004年08期

4 周紅蕾;李春玲;王貴平;侯加法;;大豆中抗?fàn)I養(yǎng)因子及其去除方法概述[J];飼料工業(yè);2006年03期

5 梅俊杰;;稀有密碼子對大腸埃希菌表達(dá)外源蛋白的影響[J];國外醫(yī)學(xué)(微生物學(xué)分冊);1999年01期

6 朱紅裕;李強;;外源蛋白在大腸桿菌中的可溶性表達(dá)策略[J];過程工程學(xué)報;2006年01期

7 夏海鋒;張顯;金雄華;劉婷婷;鄭志永;饒志明;;鎳離子親和層析介質(zhì)的制備及其用于組氨酸標(biāo)記蛋白質(zhì)的純化[J];江南大學(xué)學(xué)報(自然科學(xué)版);2010年06期

8 郭廣君;呂素芳;王榮富;;外源基因表達(dá)系統(tǒng)的研究進(jìn)展[J];科學(xué)技術(shù)與工程;2006年05期

9 向文勝,張文吉,王相晶,覃兆海;EPSP合成酶的特性及新抑制劑的研究進(jìn)展[J];農(nóng)藥學(xué)學(xué)報;2000年02期

10 廖美德,謝秋玲,林劍,洪岸,孫奮勇;外源基因在大腸桿菌中的高效表達(dá)[J];生命科學(xué);2002年05期

，

本文編號：1850048