转鲑鱼降钙素基因酵母的发酵条件优化及安全性评价
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摘要
降钙素是一种由32个氨基酸组成的多肽类激素,其蛋白结构的N端为二硫键,C端为脯氨酰胺基团。它在哺乳动物中起源于甲状腺C细胞,在鱼类中后腮体分泌。现在临床上广泛使用的是合成的人、鲑鱼降钙素,天然来源的猪降钙素和鳗鱼降钙素类似物,主要用于骨质疏松、高钙血症、变形性骨炎及类风湿性关节炎的治疗。由本实验室姜勇构建的转鲑鱼降钙素基因酵母yAGA2-r-sCT经实验证明具体内生物活性,这为开发鲑鱼降钙素的口服途径创造了可能,在生产上具有较大的应用潜力。为了提高发酵产量,我们对转鲑鱼降钙素基因酵母的发酵条件进行了优化,并通过动物毒理学实验对其进行了安全性评价,为实现降钙素的口服应用提供了实验数据。
转鲑鱼降钙素基因酵母发酵条件的优化包括对实验用酵母培养基的改良、外界培养条件的优化、工业用培养基组成及其诱导条件的优化：(1)以豆粕水解液作为氮源,采用正交设计方法,得出转基因酵母改良增殖培养基配方为：6%豆粕水解液,3%葡萄糖,0.5%酵母提取物,1%蛋白胨,pH 5。(2)通过单因素实验,对影响酵母生长的外界条件包括温度、发酵时间、接种量、搅拌转速和通气量进行优化,得出优化条件为：温度28℃,搅拌转速为200 rpm,通气量为8 L/min,接种量为8%,培养时间为3天。(3)采用部分因子设计筛选出显著影响转基因酵母生长的因子为豆粕水解液、葡萄糖和NaCl,并利用最速上升实验确定了这三个因子接近最大响应值所在的区域,最后结合响应面分析法得到了转基因酵母工业用增殖培养基最佳优化组合为：86.5g/L豆粕水解液,56.8g/L葡萄糖,1.10 g/L NaCl,5.0g/L K2HPO4,0.6 g/L (NH4)2SO4,0.2g/L MgSO4。(4)采用流式细胞和ELISA检测,通过对影响酿酒酵母表达鲑鱼降钙素蛋白的多个因素,包括诱导起始浓度、诱导时间、半乳糖浓度和添加方式的研究,结果表明：转鲑鱼降钙素基因酵母采用摇瓶发酵培养时,起始OD为0.8,诱导时间为12 h,半乳糖浓度为2%且一次性添加的方式下,外源蛋白的表达量达到5.92%。
以大鼠和小鼠为实验对象对表达鲑鱼降钙素的转基因酵母进行了毒理学实验,包括急性毒性实验、遗传毒性实验(小鼠骨髓微核实验、小鼠精子畸形实验)传统致畸实验、大鼠30天喂养实验和长期毒性实验。经口急性毒性实验显示LD50>10.0g/k,表明转基因酵母属于实际无毒物质；遗传毒性实验结果显示小鼠红细胞微核率和精子畸形率与对照组相比没有显著差异,且无剂量-效应关系,表明转基因酵母不具有遗传毒性,没有致突变作用；传统致畸实验结果显示灌胃转基因酵母的孕鼠生长发育良好,对所产小鼠胚胎外观和胎鼠内脏和骨骼观察无致畸现象,表明转基因酵母没有对孕鼠产生母体毒性,对母鼠的生殖机能没有不良影响,对小鼠胚胎发育无致畸作用；30天喂养实验中大鼠增重、摄食量、血液和血液生化指标、脏器系数均在正常范围内,与对照组均无显著差异(P>0.05),脏器组织切片检查未发现病理性变化；90天长期毒性实验结果显示,长期饲喂转鲑鱼降钙素基因酵母不会对实验动物产生毒副作用,最大无作用剂量在5.0 g/kg·BW以上,相当于人体推荐剂量0.8 g/kg·BW.因此转鲑鱼降钙素基因酵母未对实验动物造成不良影响,是安全无毒的。
Calcitonin (CT), a polypeptide hormone consisting of 32 amino acids with an N-terminal disulfide bridge and a C-terminal prolineamide residue. It is origined from the thyroid C cells in mammals and secreted from ultimobranchial body in fish. Four forms of CT are used clinically, namely synthetic human CT (hCT), synthetic salmon CT (sCT), natural porcine CT (pCT), and a synthetic analogue of eel CT. They are widely utilized as bone resorption inhibitors for the treatment of osteoporosis, hypercalcemia, Paget's disease, and rheumatoid arthritis. Recombinant saccharomyces cerevisiae expressing salmon calcitonin, namely yAGA3-r-sCT, is showed the activity in the experiments in vivo by Jiang Yong and it makes a chance for oral absorption delivery system of calcitonin. Optimization research of fermentation for yAGA3-r-sCT is carried out to obtain high production and safety evaluation is necessary which is performed by a series of toxicological experiments. The results will supply the experimental data for application of CT by oral absorption..
Optimization of fermentation for yAGA2-r-sCT includes several parts as follows. Orthogonal design is used to improve primary medium composition for production used in the lab, and the optimal medium composition is obtained as follws:6% defatted soybean hydrolysate (DSH),3%glucose,0.5%yeast extract,1%peptone, pH 5. One variable at a time design is used to optimize environment factors effecting on growth of yAGA2-r-sCT, and the optimal condition is obtained as follows: temperature 28℃, agitation 200 rpm, aeration 8 L/min, Inoculum volume8%, fermentation time 3 days. Fractional factorial design is used in the first step to evaluate the effects of 6 variables on industrial medium composition and DSH, glucose, NaCl are screened as significant factors. Then, their response region close to the maximum production is obtained by steepest ascent design. Their concentrations are further optimized using response surface methodology based on central composite design. The optimum predicted medium for maximum production of recombinant sacchromyces cerevisiae yAGA2-r-sCT is found to comprise:86.5 g/L DSH,56.8 g/L glucose,1.10 g/L NaCl,5.0 g/L K2HPO4,0.6 g/L (NH4)2SO4,0.2 g/L MgSO4. At the end, Optimization of fermentation for inducing expression of sCT is found to comprise:initial OD600 0.8, induction time 12 h, galactose 2% added at one time. The maximum expression of sCT is 5.92%.
To evaluate the toxicological safety of recombinant sacchromyces cerevisiae yAGA2-r-sCT, tests include acute toxicity test, genetoxic tests (bone marrow cell erythrocyte micronucleus test, sperm shape abnormality test), traditional teratogenicity test,30-days feeding test, and long-term toxicity test for 90 days are performed. Results:yAGA2-r-sCT has no acute toxicity (LD50>10.0 g/kg).The results of genetoxic tests shows that the micronucleus rate and sperm shape abnormality rate are not significantly different compared with the control, so yAGA2-r-sCT has no genetoxicity or nutation. (P>0.05). The appearance, viscera, skeleton of fetal mice and reproduction of female mice are not affected by yAGA3-r-sCT, so there are no maternal toxicity or embryotoxicity in teratogeniccity test. No significant toxicity is detected in 30 days feeding test and long-term toxicity test for 90 days. The maximum tolerated oral dose of yAGA2-r-sCT is more than 5.0 g/kg-BW in mice while human recommended dose is 0.8 g/kg·BW. Conclusion: recombinant Saccharomyces cerevisiae expressing salmon calcitonin is safe and nontoxic.
转鲑鱼降钙素基因酵母发酵条件的优化包括对实验用酵母培养基的改良、外界培养条件的优化、工业用培养基组成及其诱导条件的优化：(1)以豆粕水解液作为氮源,采用正交设计方法,得出转基因酵母改良增殖培养基配方为：6%豆粕水解液,3%葡萄糖,0.5%酵母提取物,1%蛋白胨,pH 5。(2)通过单因素实验,对影响酵母生长的外界条件包括温度、发酵时间、接种量、搅拌转速和通气量进行优化,得出优化条件为：温度28℃,搅拌转速为200 rpm,通气量为8 L/min,接种量为8%,培养时间为3天。(3)采用部分因子设计筛选出显著影响转基因酵母生长的因子为豆粕水解液、葡萄糖和NaCl,并利用最速上升实验确定了这三个因子接近最大响应值所在的区域,最后结合响应面分析法得到了转基因酵母工业用增殖培养基最佳优化组合为：86.5g/L豆粕水解液,56.8g/L葡萄糖,1.10 g/L NaCl,5.0g/L K2HPO4,0.6 g/L (NH4)2SO4,0.2g/L MgSO4。(4)采用流式细胞和ELISA检测,通过对影响酿酒酵母表达鲑鱼降钙素蛋白的多个因素,包括诱导起始浓度、诱导时间、半乳糖浓度和添加方式的研究,结果表明：转鲑鱼降钙素基因酵母采用摇瓶发酵培养时,起始OD为0.8,诱导时间为12 h,半乳糖浓度为2%且一次性添加的方式下,外源蛋白的表达量达到5.92%。
以大鼠和小鼠为实验对象对表达鲑鱼降钙素的转基因酵母进行了毒理学实验,包括急性毒性实验、遗传毒性实验(小鼠骨髓微核实验、小鼠精子畸形实验)传统致畸实验、大鼠30天喂养实验和长期毒性实验。经口急性毒性实验显示LD50>10.0g/k,表明转基因酵母属于实际无毒物质；遗传毒性实验结果显示小鼠红细胞微核率和精子畸形率与对照组相比没有显著差异,且无剂量-效应关系,表明转基因酵母不具有遗传毒性,没有致突变作用；传统致畸实验结果显示灌胃转基因酵母的孕鼠生长发育良好,对所产小鼠胚胎外观和胎鼠内脏和骨骼观察无致畸现象,表明转基因酵母没有对孕鼠产生母体毒性,对母鼠的生殖机能没有不良影响,对小鼠胚胎发育无致畸作用；30天喂养实验中大鼠增重、摄食量、血液和血液生化指标、脏器系数均在正常范围内,与对照组均无显著差异(P>0.05),脏器组织切片检查未发现病理性变化；90天长期毒性实验结果显示,长期饲喂转鲑鱼降钙素基因酵母不会对实验动物产生毒副作用,最大无作用剂量在5.0 g/kg·BW以上,相当于人体推荐剂量0.8 g/kg·BW.因此转鲑鱼降钙素基因酵母未对实验动物造成不良影响,是安全无毒的。
Calcitonin (CT), a polypeptide hormone consisting of 32 amino acids with an N-terminal disulfide bridge and a C-terminal prolineamide residue. It is origined from the thyroid C cells in mammals and secreted from ultimobranchial body in fish. Four forms of CT are used clinically, namely synthetic human CT (hCT), synthetic salmon CT (sCT), natural porcine CT (pCT), and a synthetic analogue of eel CT. They are widely utilized as bone resorption inhibitors for the treatment of osteoporosis, hypercalcemia, Paget's disease, and rheumatoid arthritis. Recombinant saccharomyces cerevisiae expressing salmon calcitonin, namely yAGA3-r-sCT, is showed the activity in the experiments in vivo by Jiang Yong and it makes a chance for oral absorption delivery system of calcitonin. Optimization research of fermentation for yAGA3-r-sCT is carried out to obtain high production and safety evaluation is necessary which is performed by a series of toxicological experiments. The results will supply the experimental data for application of CT by oral absorption..
Optimization of fermentation for yAGA2-r-sCT includes several parts as follows. Orthogonal design is used to improve primary medium composition for production used in the lab, and the optimal medium composition is obtained as follws:6% defatted soybean hydrolysate (DSH),3%glucose,0.5%yeast extract,1%peptone, pH 5. One variable at a time design is used to optimize environment factors effecting on growth of yAGA2-r-sCT, and the optimal condition is obtained as follows: temperature 28℃, agitation 200 rpm, aeration 8 L/min, Inoculum volume8%, fermentation time 3 days. Fractional factorial design is used in the first step to evaluate the effects of 6 variables on industrial medium composition and DSH, glucose, NaCl are screened as significant factors. Then, their response region close to the maximum production is obtained by steepest ascent design. Their concentrations are further optimized using response surface methodology based on central composite design. The optimum predicted medium for maximum production of recombinant sacchromyces cerevisiae yAGA2-r-sCT is found to comprise:86.5 g/L DSH,56.8 g/L glucose,1.10 g/L NaCl,5.0 g/L K2HPO4,0.6 g/L (NH4)2SO4,0.2 g/L MgSO4. At the end, Optimization of fermentation for inducing expression of sCT is found to comprise:initial OD600 0.8, induction time 12 h, galactose 2% added at one time. The maximum expression of sCT is 5.92%.
To evaluate the toxicological safety of recombinant sacchromyces cerevisiae yAGA2-r-sCT, tests include acute toxicity test, genetoxic tests (bone marrow cell erythrocyte micronucleus test, sperm shape abnormality test), traditional teratogenicity test,30-days feeding test, and long-term toxicity test for 90 days are performed. Results:yAGA2-r-sCT has no acute toxicity (LD50>10.0 g/kg).The results of genetoxic tests shows that the micronucleus rate and sperm shape abnormality rate are not significantly different compared with the control, so yAGA2-r-sCT has no genetoxicity or nutation. (P>0.05). The appearance, viscera, skeleton of fetal mice and reproduction of female mice are not affected by yAGA3-r-sCT, so there are no maternal toxicity or embryotoxicity in teratogeniccity test. No significant toxicity is detected in 30 days feeding test and long-term toxicity test for 90 days. The maximum tolerated oral dose of yAGA2-r-sCT is more than 5.0 g/kg-BW in mice while human recommended dose is 0.8 g/kg·BW. Conclusion: recombinant Saccharomyces cerevisiae expressing salmon calcitonin is safe and nontoxic.
引文
鲍清,安丽.畸形精子的分型与观察.卫生毒理学杂志,2004,18(4)：305
曹芙萍.降钙素药理与临床应用进展.安徽医药,2006,10(10)：777～779
曹佳主编.微核试验.北京：军事医学科学出版社.2000.264～272
陈敏,王静馨.模糊正变法用于锌酵母发酵培养基条件优化的研究.食品与发酵工业,1994,(5)：24～28
陈卫,陆英,田丰伟,等.干酪乳杆菌LC-15的增菌培养基设计及生长动力学研究.食品与发酵工业,2008,34(8)：1～5
褚以文.微生物培养基优化方法及其OPTI优化软件.国外医药抗生素杂志分册,1999,20(2)：58～61
陈锡文,邓楠.中国食品安全战略研究.北京：化学工业出版社,2004
狄旭,陈松森,宋飞,等.鲑鱼降钙素基因的克隆及其在大肠杆菌中的表达.中国医学科学院学报,1996,18(6)：423～428
丁轲,程安春.乳酸杆菌表达系统的研究进展.中国畜牧兽医学会动物微生态学分会第三界第七次学术研讨会论文集,2004,45～49
丁绍峰,谭天伟.豆粕水解液为氮源细菌厌氧流加发酵生产L-乳酸.过程工程学报,2006,6(1)：77～81
董丹,刘昱辉,吴元华.基因工程法生产降钙素的研究进展.沈阳药科大学学报,2006,23(8)：547～552
窦毅芳,刘飞,张为华.响应面建模方法的比较分析.工程设计学报,2007,14(5)：359～363
杜清友,赵明,王会信,等.鲑鱼降钙素基因在大肠杆菌中的克隆与表达.生物化学杂志,1997,13(5)：531～536
付仲文,连庆,李宁.转基因植物食用安全性评价现状.农业科技理,2009,28(6)：24-27,37
高英茂.组织学与胚胎学彩色图谱和纲要.北京：科学出版社.2005
郭世绂.骨质疏松治疗的新进展.现代诊断与治疗,2003,14(5)：257～62
何召庆,秦泽荣,徐镔蕊,等.降钙素基因在乳酸杆菌中的克隆.中国兽药杂志,2002,36(6)： 20～22
黄建华,邱然,杨文洲,等.金属离子对啤酒酵母发酵中生成乳酸的影响.食品技术,2007(7)：123-124
黄丽金,陆兆新,袁勇军.响应面法优化德氏乳杆菌保加利亚亚种增殖培养基.食品科学,2005,26(5)：103～106
黄迎春,何壁梅,张义正,等.鲑鱼降钙素串联基因的克隆表达及重组蛋白的生物学活性.生物技术,2003,13(6)：8～10
姬华,乐国伟,王洪新,等.转基因食品的营养学评价研究进展.食品研究与开发,2009,30(7)：149～152
姜勇.鲑鱼降钙素在酵母中的多拷贝表达及安全性评价[博士学位论文].青岛：中国海洋大学,2008
姜勇,张学成,陈云松,等.rDNA介导的酵母表达鲑鱼降钙素基因多拷贝整合载体的构建.中国海洋大学学报,2009,39(3)：443～447
金汝城.均匀设计和正交设计在黄芪活性成份提取中的应用比较.生物数学学报,2009,24(3)：518-522
李国华,席小平,贾继峰,等.Wistar大鼠主要脏器系数与体重相关性的讨论.山西医药杂志,2005,34：661～662
李宏.微核试验的研究进展.安徽农业科学,2009,37(7)：2864～2866
李来好,郝志明,吴燕燕.基因食品的营养安全问题.现代食品科技,2005,21(2)：166～170
李宁.国内食品安全性毒理学评价的现状和发展.毒理学志,2007,21(5)：368～370
李宁.转基因食品的食用安全性评价.毒理学杂志,2005,6(19)：163～165
李寿祺,主编.卫生毒理学基本原理和方法.成都：四川科学技术出版社,1987,82
李兆鹏,张栩,徐斌.重组大肠杆菌高密度发酵中乳糖诱导表达hBLyS.过程工程学报,2005,5(4)：446～449
李岩,高虹,孙井江.Wistar大鼠体重和主要脏器参考值的研究.实用预防医学,2009,16(6)：1708～1711
李悠慧.我国食品毒理学发展50年.中国食品卫生杂志,1999,11(6)：5～6
李永仙,郑飞云,李崎.RSM法优化产p-葡聚糖酶的发酵培养基.食品工业科技,2009,30(8)：137-142
联合国粮农组织.食品安全风险分析(,樊永祥).北京：人民卫生出版社,2008
刘建中,熊亚红,翁丽萍,等.生物过程的优化.中山大学学报,2002,41：134～137
刘恺,张占吉,牛树启.转基因食品安全性评价综述.保定师范专科学校学报,2006,19(4)：18～20
刘深基,陈松森,张松,等.鲑鱼降钙素基因在大肠杆菌中的可溶性高效表达.基础医学与临床,1999,19(2)：89～93
刘向勇,张小华,鲍晓明.酒酵母工业菌株胁迫条件耐受性分析.中国酿造,2006(1)：8～11
刘颖,梁东春,郭刚,等.鲑鱼降钙素重组杆状病毒载体的构建及表达.现代口腔医学杂志,2003,17(1)：14～17
刘毓谷.卫生毒理学基础.北京：人民卫生出版社,1995
付立杰.现代毒理学及其应用.上海：上海科学技术出版社,2001
马复兴,邹复海.降钙素.中国医药学杂志,1983,3(3)：18～20
孟迅吾,周学瀛.骨质疏松症的诊断、预防和治疗.中华内分泌代谢杂志,1993,9(4)：246～248
潘良文.主要商业化转基因产品标准化检测技术的建立与应用[博士学位论文].南京：南京农业大学,2004
沈娟,陆兆新,别小妹,等.Bacillus sp.fmbJ224发酵产新型抗菌肽培养基的优化研究.生物工程学报,2005,4(21)：609～614
沈倩,胡德文,时春.神经网络应用技术.长沙：国防大学出版社,1993
史志诚.动物毒物学.北京：中国农业出版社,2001,6月,第一版
宋晓金.富含DHA的裂殖壶菌的工业化生产试验-脂肪酸提取及应用研究：[博士学位论文].青岛：中国海洋大学,2008
孙金旭,朱会霞.响应面法优化灰树花发酵培养条件研究.中国酿造,2009,9(210)：94～98
孙平楠.酵母表达鲑鱼降钙素基因载体的构建及相关研究[博士学位论文].青岛：中国海洋大学,2007
隋海霞,高艽,耿桂英,等.鹿茸人参制剂的毒理学安全性评价.现代预防医学,2007,34(23)：4448～4452
唐启义,冯明光.实用统计分析及其DPS数据处理系统.北京：科学出版社,2002
万琦,陆兆新.脱苦大豆多肽产生菌的筛选及其水解条件的优化.食品科学,2003,24(2)：29～32
王继花,薛永常,高圣.高渗胁迫下啤酒酵母的生理特性研究.中国酿造,2008,(45)：45～47
王普,孙立明,何军邀.响应面法优化热带假丝酵母104菌株产羰基还原酶发酵培养基.生物工程学报.2009,25(6)：863～868
王迁.欧美转基因食品法律管制制度比较研究.河北法学,2005,23(10)：114～121
王如平,马玉银.转基因产品的安全性评价.安徽农业科学,2008,36(36)：16097～16098,16139
王翔林,梁践,孙玉昆.降钙索基因工程研究.沈阳药学院学报,1995,12(4)：258～262
王晓辉,察少华,迟乃玉,等.海洋低温BS070623菌株选育及其发酵培养基优化(Ⅰ).渤海大学学报,2009,30(2)：97～100
王亚东,杨海燕,王海玉,等.SD大鼠30 d喂养试验正常参考值的探讨.现代预防医学,2007,34(7)：1265～1267
王忠华,周美园,夏英武.转基因产品的食用安全性评价.生命科学,2002,14(5)：305～307
文剑明,郑铭豪.破骨细胞的形成功能及细胞因子调节.中国骨质疏松杂志,1998,41：64～68
吴风霞,李纯厚,戴明.人工神经网络在海洋科学中的应用.南方水产,2009,5(1)：75～80
吴梧桐.生物制药工艺学.北京：中国医药科技出版社,1993.1-642
吴小平,孙洁胤.均匀设计在药学中的应用.浙江省医学科学院学报,2007,71：38～40
项华,章晓玲,查捷,等.SD大鼠生长发育指标和主要脏器正常参考值的探讨.中国卫生检验杂志,2008,18(2)：299～301
肖怀秋,李玉珍.微生物培养基优化方法研究进展.酿酒科技,2010(1)：90～94
谢恩C.加德.药物安全性评价.北京：化学工业出版社,2006：106～140
谢南.丙烯酰胺对雄性小鼠生殖系统毒性的研究[硕士学位论文].吉林：2007
杨冠珍,陈珍珍,崔大敷,等.人修饰型降钙素和鼠酰胺化酶基因在昆虫细胞中的偶连表达.生物工程学报,2002,18(1)：20～24
杨继良,项时康,杨伟华,等.棉子饼粉制取氨基酸水解液的研究.中国棉花,1995,22(4)： 8～10
杨永涛,涂国全.响应面法优化弗氏链霉菌S-221变种发酵培养基.生物加工过程,2005,3(2)：36～40
于峰,姚宝玉,王薏,等.小鼠畸形精子分型.上海实验动物科学,1997,17：232～233
余明泽,刘以农,蒋中仁等.环磷酰胺诱导小鼠骨髓细胞微核的影响因素的研究.现代预防医学,2007,34(5)：935～941
袁伯俊,廖明阳,李波.药物毒理学实验方法与技术.北京：化学工业出版社,2007.1～368
袁志发,周静芋.试验设计与分析(第二版).北京：高等教育出版社,2000.1～476
张虹,袁红梅.转基因食品安全性评价与管理.高师理科刊,2008,28(2)：71～74
张宏灿.豆饼酸水解制氨基酸复合液的研究.食品科学,1998,19(11)：59～61
张健,李夏明,汪仁官.两水平无重复析因设计的一个散度效应检验方法.工程数学学报,2006,23(4)：705～708
张桥,主编.卫生毒理学基础.北京：人民卫生出版社,2000.244～315
张军明.组织学与胚胎学实用彩色图谱.浙江：浙江大学出版社,2004.1～103
张希桥,杨志华.关于精子畸变分型的参考意见.卫生毒理学杂志.1995,9：46
张铣,刘毓谷.毒理学.北京：北京医科大学-中国协和医科大学联合出版社,1997.1～300
张玉彬,吴梧桐,吴文俊.降钙素的研究进展.中国医药学杂志,1996,31(8)：451～454
张绪金,唐英华.降钙素进展.中国骨质疏松杂志,2004,10(1)：96～97
曾北危.转基因生物安全.北京：化学工业出版社,2004.10～26
赵丽坤,郭会灿.微生物培养基优化方法概述,2008,20(4)：50～52
赵吉强,李霞,程智慧,等.鲑鱼降钙素基因向烟草遗传转化的初步研究.西北农林科技大学学报,2002,30：61～64
赵吉强,李霞,李丽霞,等.生菜遗传转化受体系统的建立及鲑鱼降钙素基因的导入.烟台大学大学学报(自然科学与工程版),2004,17(2)：116～121
郑定仙.我国食品毒理学回顾与展望.中国热带医学,2006,6(10)：1880～1881
郑莉,赵明,王会信.人降钙素基因在大肠杆菌中的克隆与表达.军事医学科学院院刊,1998,22(1)：23-26
中华人民共和国卫生部.GB15193.21-2003.食品安全性毒理学评价程序和方法.北京：中国标准出版社,2003
中华人民共和国卫生部.保健食品检验与评价规范(2003年版)(卫生部文件：卫法监发 (2003)42号).京,2003
中华人民共和国卫生部.食品安全性毒理学评价程序和方法(试行).北京：中国标准出版社,1985
中华人民共和国卫生部.GB15193-1-15193.1994.食品安全性毒理学评价程序和方法.北京：中国标准出版社,1995.56～57
中华人民共和国卫生部第28号令.转基因食品卫生管理办法,2001
中华人民共和国卫生部药政局.新药(西药)临床研究指导原则汇编(毒理学).1993：216～217
钟辉,彭英芳,李平,等.鲑鱼降钙素与骨生长肽融合基因的克隆及其在毕赤酵母中的表达.中国生物工程杂志,2002,22：79～82
钟为章,罗一菁,张忠智,等.红螺菌科光合细菌液体培养基的优化,2009,26(7)：67～69
周世祥,杨丽珠.试论转基因技术的安全性问题及其社会控制.成都理工大学学报(社会科学版),2006,14(1)：11～15
周一江,张学成,王玉梅,等.人/鲑降钙素嵌合基因在大肠杆菌中的串连表达.中国海洋大学学报,2007,37(1)：103～106,114
邹文安,刘宝,姜波等.基于Excel技术平台人工神经网络BP模型及应用.水文,2010,30(1)：44～46,63
GB 15193.5-2003,骨髓细胞微核试验[S].中国国家标准汇编(2003年修订-17.北京：中国标准出版社,2004,609～612
GB 15193.7-2003,小鼠精子畸形试验[S].中国国家标准汇编(2003年修订-17).北京：中国标准出版社,2004,619～622
Adow D A, Zwahlen C. Assessing environmental risks of transgenicplants. Ecology Letter,2006, 9:196～214
Anderson W A. The future relationship between the media,the food industry and the consumer. Br. Med. Bull.2000.56(1):254～268
Angela E, Kerry L.Dearfield, Michael C.Cimino. Mutagenicity test schemes and guidelines: U.S.EPA office of pollution prevention and toxics and office of pesticide programs. Environ Mole Mutagen,1993,21(1):38～45
Annadurai G. Design of optimum response surface experiments for adsorption of direct dye on chitosan. Biopronc Eng,2000,23:451～455
Arnold F H, Volkov A A. Directed evolution of biocatalysts. Curr Opin Chem Biol,1999,3: 54～59
Arntzen C J. Immunity for breakfast, Science,1998,280(8):831
Austin LA, Hunter H. Calcitonin:physiology and pathophysiology. N Engl J Med,1981,304(5): 269～276
Azria M, Copp D H, Zaneli J M.25 years of salmon calcitonin:from synthesis to therapeutic use. Calcif Tissue Int,1995,57:405
Barton J. Biotechnology for developing country agriculture-Intellectual property protection Washington, D.C:International Food Policy Research Institute,1999
Berg P, Singer M F. The recombinant DNA controversy:twenty years later. Bio/Technology. 1995,13,1132～4
Blanquet S, Marol-Bonnin S, Beyssac E, et al. The Biodrug concept:an innovative approach to therapy. Trends Biotechnol,2001,19:393～400
Blanquet S, Antonelli R, Laforet L, et al. Living recombinant Saccharomyces cerevisiae secreting proteins or peptides as a new drug delivery in the gut. Biotechnol,2004,110:37～49
Box GEP, Behnken DW. Some new three level designs for the study of quantitative variables. Technometrics.1960,2:455～475
Box GEP, Hunter, W.G, Hunter J S. Statistics for experiments:an introductory to design, data analysis, and model building. New Nork, John Wiley & Sons,1978
Bradbury A F, Finnie M D A, Smyth D G, et al. Mechanism of C-terminal amide formation by pituitary enzymes. Nature.1982,298:686～688
Chakraborty C, Nandi S, Sinha S. Overexpression, purification and characterization of recombinant salmon calcitonin, a therapeutic protein, in Streptomyces avermitilis. Protein Pept Lett,2004,11(2):165～173
Chambers, T. J., and Magnus, C. J. Calcitonin alters behaviour of isolated osteoclasts. J Pathol, 1982,136:97～106
Chi Z, Liu J, Zhang W. Trehalose accumulation from soluble strarch by Saccharomycopsis fibuligera sdu. Enzyme Microb TechnoL,2001,28:240～245
Chowdhury E H, Kuribara H, Sultana P. Detection of corn intrinsic and recombinant DNA fragment and Cryl Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt. Journal of animal science,2003,81:2546～2551
Ceserani R. Calcitonin and prostaglandin System. Life Sci,1979,25:1851～1856
Cockburn A. Assuring the safety of genetically modified (GM) foods:the importance of a holistic. Integrative approach Biotechnology,2003,98(1):79～106
Codex Alimentarius Commission. Joint FAO/WHO food standard programme. Chiba, Japan,2000: 14～17
Copp, D.H., Cameron, E. C., Cheney, B. A., Davidson, A. G. F., and Heinze K. G. Evidence for calcitonin-A new hormone from the parathyroid that lowers blood calcium. Endocrinology.1962 a 70:638～649
Copp, D. H. and Cheney, B. A. Calcitonin-A hormone from the parathyroid that lowers the calcium level of the blood. Nature.1962 b,193:381～382
Copp, D. H., Niall, H. D., Keutmann, H. T. and Potts J. T. Amino acid sequence of salmon ultimobranchial calcitonin. Proc Natl Acad Sci USA.1969,64:771～778
Cott MK, Vining GG, Douglas CM, et al. Modifying a central composite design to model the process mean and variance when there are hard-to-change factors. App.l Statist.2006,55: 615～630
Cranney A, Tugwell P, Zytaruk N, et al. Meta2Analysis of Calcitonin for the Treatment of Postmenopausal Osteoporosis. Endocrine Reviews,2002,23(4):540～51
Dan YE, Zhi-nan Xu and Pei-lin CEN. Medium Optimization for Enhanced Production of Cytosine-substituted Mildiomycin Analogue (MIL-C) by Streptoverticillium rimofaciens ZJU 5119. J Zhe-jiang Univ Sci B,2008,9(1):1581～1673
Daniel Heller. A Review and Applications of Flow Cytometry.2004,1-13
Day A, Meyling J C. Practical experience with rapid methods of detecting yeasts in breweries. J Inst Brew,1990,89:204～206
Desai K M, Akolkar S K, Bae Y P, et al. Optimization of fermentation media for exopoly saccharide production from Lactobocillue plantarum using artificial intelligence-based tech-niques. ProcessBiochemistry,2006,41:1842～184
Eipper B A, Perkins S N, Husten E J, et al. Peptidyl-α-hydroxyglycine|α-amidatinglyase. J Biol Chem,1991,266:7827-7833
Ellstand N C, Prentice H C, Hancock J F. Gene flow and introgression from domesticated plants into their wild relatives. Annual Review Of Ecology and Systematics,1999,30:539～563
Ellstand N C. Current knowledge of gene flow in plants:Implications for transgene flow. Philosophical Transactions of the Royal Society B. Biological Sciences,2003,358:1163-1170
Ertola R J, Giulietti AM, Castillo F J. Design, Formulation and Optimization of Media. Bioprocess Technol,1995,21:89～137
Ewen S W, Pusztai A. Health risks of genetically modified food. Lancet,1999,354:684～690
EU. Commission of the European Communities. Communication from the commission on the precautionary principle COM 1. Brussels.2000
Fang KT, Winker P. Uniform design. Theory and Applications. Technometrics.2000,42:237～248 FAO/WHO. Biotechnology and food safety. RomeL Report of Second joint FAO/WHO Consulta-tion,1996
FAO/WHO. Evaluation of allergenicity of genetically modifiedfood. Report of a joint FAO/WHO expert consultation on allergenicity of food derived from biotechnology. FAO, Rome,2001
FAO/WHO. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechno-logy, Safety Aspects of Genetically Modified Foods of Plant Origin. World Health Organisation, Publication details,2000
Fenech M. The cytokinesis-block micronucleus technique:A detailed description of the method and its application to genotoxicity studies in human populations. Mutation Res,1993,285:35～44
Feng Y, He Z, Ong SL. Optimization of agitation, aeration, and temperature conditions for maximum β-mannanse production. Enzyme Microb TechnoL,2003,32:282～289
Foster G V, Baghdiantz A, Kumar M A, Slack E, Soliman H A and MacIntyre I. Thyroid origin of calcitonin. Nature,1964,202:1303～1305
Foster GV, MacIntyre I, and Pearse AGE. Calcitonin production and the mitochondrion-rich cells of the dog thyroid. Nature,1964,203:1029～1030
Foy CA, Parkes HC. Emerging homogeneous DNA-based technologies in the clinical laboratory. Clin Chem,2001,47(6):990～1000
Franco-Lara E, Link H, Weuster-Botz D. Evaluation of artificial neural networks for modeling and optimization of medium composition with a genetic algorithm. Process Biochemistry,2006,41: 2200～2206
Gennari C. Dolore osseo. Endorfine calcitonine. In:Gennari C, Segre G. eds. The effects of calcitonin in man. Proc.lst int. Wkshp. Florence 1982. Masson, Milan1983.213～222
George A S. Challenges for methods to detect genetically modified DNA in foods. Food Control, 1999,10:351～352
Grillot-courvalin C, Goussard S, Huetz F, et al. Functional gene transfer from intracellular bacteria to mammalian cells. Nature Biotechnol,1998,16:1～5
Hai-feng Pan, Wen-na Bao, Zhi-peng Xie. Optimization of medium composition for cisepoxysuccinate hydrolase production in Escherichia coli by response surface methodology. African Journal of Biotechnology,2010,9(9):1366～1373
Heddle J A. A rapid in vivo test for chromosomal damage. Mutal Res,1973,18:187～190
Henriksen DB, Rolland M, Jakobsen MH, et al. C-terminal amidation of calcitonin by carboxype-ptidase Y catalyzed transpeptidation with a photocleavable nucleophile. Pep Res,1992,5(6):321-342
Hiatt A, Cafferkey R, Bowdish B. Production of antibodies in transgenetic plants. Nature,1989, 342:76～78
Hirsch PF, Gauthier GF, Munson PL. Thyroid hypocalcaemic principle and recurrent laryngeal nerve injury as factors affecting the response to parathyroidectomy in rats. Endocrinology,1963, 73:244～250
Hohmann S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev, 2002,66(2):300～372
Huppatz F L. Fitzgerald P A. Genetically-modified foods-safety and regulatory issues. J. Aust. 2000,172(4):170～173
Isar J, Agarwal L, Saran S, et al. A statistical approach to study the interactive effects of process parameters on succinic acid production from Bacteroidesfragilis. Anaerobe,2007,13:50～56
Ishikawa J, Kawaguchi J, Tamaoki H. Production of human calcitonin as tandemly repeat fusion protein. JP05320196,1993-13-3
Kishor CU, Kamlesh CP. Statistical screening ofmedium components by Plackett-Burman design for lactic acid production byLactobacillussp. KCP 01 using date juice. Bioresource Technology, 2007,98:98～103
Klotz A, Mayer J, Einspanier R. Degradation and possible carry over of feed DNA monitiored in pigs and poultry. Food Rearch Technology,2002,214:271～275
Koji Satake, Toshiaki Monaka, Satoshi Yamada et al. The rapid development of diesel engines using an optimization of the fuel injection Control. Mitsubishi Heavy Industries,2008,45:6-10
Kowalchuk G A, Bruinsma M, Van Veen J A. Assessing responses of soil microorganisms to GM plants. Trends in Ecology and Evolution,2003,18:403～410
Kuiper H A, Kleter G A, Kok E J.Assessment of the food safety issues related to genetically modified food. Plant,2001,27(6):503～528
Kumar MA, Foster GV, MacIntyre I. Further evidence for calcitonin:A rapid acting hormone which lowers plasma calcium. Lancet ii,1963:480～482
Leeder S R. Genetically modified foods-food for thought. Med. J. Aust.2000,172(4):173～174
Lems WF, Dijkmans BA. Effect van anti-osteoporotic medicine on het on tstaan van wervelfracyuren. Ned Tijdschr Geneeskd,2000,144:1941～1945
Liu C, Ruan H, Shen H, et al. Optimization of the Fermentation Medium for Alpha-galactosidase Production from Aspergillus Foetidus ZU-G1 Using Response Surface Methodology. J Food Sci, 2007,72(4):120～125
Losey J E. Transgenic pollen harms monarch larvae. Nature,1999, (399):214
Lu BR, Snow AA. Gene flow from genetically modified rice and its environmental consequences. Bioscience,2005,55:669～678
Rajasimman M, Subathra S. Optimization of Gentamicin Production:Comparison of ANN and RSM Techniques. International Journal of Chemical and Biomolecular Engineering,2009,14～19
MacIntyre I, Parsons J A, Robinson C J. The effect of thyrocalcitonin on blood bone calcium equilibrium in the perfused tibiae of the cat. J Physiol,1967,191:393～405
Mahony J, Chong S, Jang D, et al. Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chamydia trachomatis nucleic acid by PCR, ligase chain reation, and transcription-mediated amplification:identification of urinary substances associated with inhibition and removal of inhibitory activity. J Clin Microbiol,1998,36:3122～3126
Marrazzo JM, Handsfield HH, Whittington WL. Predicting chlamydial and gonococcal cervical infection:implications for management of cervicits. Obstet Gynecol,2002,100(3):579～584
Marrs B, Delagrave S, Murphy D. Novel approaches for discovering industrial enzymes. Curr Opin Microbiol,1999,2:241～245
Mason HS, Ball J M, Shij j. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immuno genicity in mice. Pro Natl Acad Sci USA,1996,93:5335～5340
Metcalfe D D, Astwood J D, Townsend R, et al. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit Rew Food Sci Nutr,1996,36(S):165～186
Millstone E, Brunner E, Mayer S. Beyond"substantial equivalence". Nature,1999,401:525～526
Mironova R, Philipova D, Marekov L, et al. Secretion of oligomeric Val8-human calcitonin by Saccharomyces cerevisiae. FEMS Microbiol Lett,1991,83:23～28
Mosely B E. Safety assessing and public concern for genetically modified food Products:The European view. Toxicol Pathol,2002,30(1):129～131
Muralidhar R V, Chirumamila R R, Marchant R, et al. A response surface approach for the comparison of lipase production by Candida cylindracea using two different carbon source. Biochem Eng J,2001,9:17～23
Neher R, Riniker B, Maier R, Byfield PGH, Gudmundsson TV, MacIntyre I. Human calcitonin. Nature,1968,220:984～986
Nuti R, Vatimmo A, Martini G, et al. Carbocalcitonin treatmenl in Sudeck's atrophy. Clin Orthop Rel Res,1987,215:217-222
OECD. Food Safety Evaluation. Paris:organization for Economic Cooperation and Development. 1996
OECD. Safety evaluation of foods produced by modern biotechnology:concerps and principles. Paris:DECD,1993:1～16
OECD. Report of the OECD workshop on the toxicological and nutritional testing of novel foods, Aussois, France,5-8 March 1997. Paris:organization for Economic Cooperation and Developme-nt.1998
OECD Consensus Doeuments. Paris:orgnisation for Economic Cooperation and Development, Inter-Agency Network for Safety in Biotechnology.2001 a
OECD. Report of the OECD workshop on Nutritional Assessment of Novel Foods and Feeds, Ottawa, Canada,5-7 February 2000. Paris:orgnisation for Economic Cooperation And Development.2001 b
Ofoghil H, Moazami] N, Domonsky N N, et al. Cloning and expression of human calcitonin genes in transgenic potato plants. Biotechnology Letters,2000,22:611～615
Parsell A D, Sauer R T. The structural stability of a protein is an important determinant in Escherichia coli. Biol Chemistry,1989,264:7590～7596
Potts J T, Niall H D, Keutmann H T, Brewer, H B, Deftos L J. The amino acid sequence of porcine thyrocalcitonin. Proc Natl Acad Sci,1968,59:1321～1328
Ray M V, Duyne P V, Bertelsen A H, et al. Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide. Biotechnology (NY),1993, 11(1):64～70
Ray M V, Meenan C P, Consalvo A P, et al. Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli. Protein Expr Purif,2002,2S(2):249～259
Ray M V, Van Duyne P, Bertelsen A H, et al. Production of recombinant salmon calcitonin by in vit ro amidation of an E. coli. produced precursor peptide. Biotechnology,1993,11(1):64～70
Reddy PRM, Reddy G, Seenayya G. Production of thermostable pullulanase by Clostridium the rmosulfuro genes SV2 in solid-state fermentation:optimization of nutrients levels using response surface methodology. Bioproc Eng,1999,21:497～503.
Reginster JY. Treatment of bone in elderly subjects:calcium, vitamin D, fluor, bisphosphonates, calcitonin. Horm Res,1995,43:83～88
Rittel W, Brugger M, Kamber B, Riniker B, Sieber P. Thyrocalcitonin.Ⅲ. Die Syntheses des alpha-thyrocalcitonins. Helv Chim Acta,1968,51:924～928
Roukas T, LiakopouLou-Kyriakides M. Production of pullulan from beet molasses by Aureobasi-dium pullulans in a stirred tank fermentor. J Food Eng,1999,40:89～94
Ruth E. Montes, Grady Hanrahan, Frank A. Gomez. Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary Electrophoresis, 2008,29:3325～3332
Sato N, Mizumoto K, Makamura M, et al. A possible role for centrosome overduplication in radiation induced cell death. Oncogene,2000,19:5281～5290
Saudagar P S. Singhal R S. Optimization of nutritional requirements and feeding strategies for clavulanic acid production by streptomyces clavuligerus. Bioresour Technol,2007,98(10):2010-2017
Shadrack R morphuti. Cartagena protocol on biosafety to the convention biological diversity. Registrar:GMO,2000
Simth N. Seeds of opportunity and assessment of the benefits safety and oversight of plant genomics and agricultural biotechnology. Committee Review of Entomology,1996, (11): 106～111
Sofuni T. Japanese guidelines for mutagenicity testing. Environ Mole Mutagen,1993,21(1):2-7
Stewart C N, Halfhill M D, Warwick S I. Transgene introgression from genetically modified crops to their wild relative. Nature Reviews of Genetics,2003,4:806～817
Sun Ping-nan, Zhang Xue-cheng, Zang Xiao-nan, et al. Anti-hypercalcemic effect of orally adm-inistered recombinant Saccharomyces cerevisiae expressing salmon calcitonin on hypercalcemic rats. Biotechnol Lett.2007,29(7):1013-1018
Tacket CO, Mason HC, Losonsky G, et al. Immunogenicity in human so far recombinant bacterial antigen deliver edina transgenic potato. Nature Medicine,1998, (14)5:607～609
Thompson D R. Response surface experimentation. J Food Proc Pres,1982, (6):155～188
Tushar Gulati, Mainak Chakrabarti, Anshu Singh, et al. RSM and ANN/GA for Optimization of Soybean. Food Technol. Biotechnol.2010,48(1):11～18
Tuysuz B, Mercimek S, Unger S, et al. Calcitonin treatment in osteoectasia with hyper-phosphas-i (juvenile Paget's diaease):Radiographic change after treatment. Pediatr Radiol,1999,29: 838～841
Wang F Q, Gao C J, Yang C Y. Optimization of all Ethanol Production Medium in very High Gravity Fermentation. Biotechnol Lett,2007,29(2):233～236
Watkinson A R, Freckleton R P, Robinson R A, et al. Predictions of biodiversity response to gene-tically modified herbicide-tolerant crops. Science,2000,289:1554～1557
Waynforth B.H. Injection techniques experimental and surgical techniques in the rat. London: Academic Press,1980.3～61
Wyrobek A J, Bruce W R. Chemical induction of sperm abnormalities in mice. Proc. Nat. Acad. Sci. USA,1975,72(11):4425～4429
Xu CP, Yun JW. Optimization of Submerged-culture Sonditions for Mycelial Growth and Exobiopolymer Production by Auricularia Polytricha (wood ears fungus) using the Methods of Uniform Design and Regression Analysis. Biotechnol Appl Biochem,2003,38:193～199
Yatuba M, Suzuki Y, Ohsuye K, et al. High expression of a recombinant human calcitonin precu-rsor peptide in Escherichia coli. Appl Microbiol Biotechnol,1995,42(5):703～708
Zaidi M, Fuller K, Bevis PJR, Gaines Das RE, Chambers TJ, MacIntyre Ⅰ. Calcitonin gene-related peptide inhibits osteoclastic bone resorption:A comparative study. Calcif Tissue Int,1987,40: 149～154
曹芙萍.降钙素药理与临床应用进展.安徽医药,2006,10(10)：777～779
曹佳主编.微核试验.北京：军事医学科学出版社.2000.264～272
陈敏,王静馨.模糊正变法用于锌酵母发酵培养基条件优化的研究.食品与发酵工业,1994,(5)：24～28
陈卫,陆英,田丰伟,等.干酪乳杆菌LC-15的增菌培养基设计及生长动力学研究.食品与发酵工业,2008,34(8)：1～5
褚以文.微生物培养基优化方法及其OPTI优化软件.国外医药抗生素杂志分册,1999,20(2)：58～61
陈锡文,邓楠.中国食品安全战略研究.北京：化学工业出版社,2004
狄旭,陈松森,宋飞,等.鲑鱼降钙素基因的克隆及其在大肠杆菌中的表达.中国医学科学院学报,1996,18(6)：423～428
丁轲,程安春.乳酸杆菌表达系统的研究进展.中国畜牧兽医学会动物微生态学分会第三界第七次学术研讨会论文集,2004,45～49
丁绍峰,谭天伟.豆粕水解液为氮源细菌厌氧流加发酵生产L-乳酸.过程工程学报,2006,6(1)：77～81
董丹,刘昱辉,吴元华.基因工程法生产降钙素的研究进展.沈阳药科大学学报,2006,23(8)：547～552
窦毅芳,刘飞,张为华.响应面建模方法的比较分析.工程设计学报,2007,14(5)：359～363
杜清友,赵明,王会信,等.鲑鱼降钙素基因在大肠杆菌中的克隆与表达.生物化学杂志,1997,13(5)：531～536
付仲文,连庆,李宁.转基因植物食用安全性评价现状.农业科技理,2009,28(6)：24-27,37
高英茂.组织学与胚胎学彩色图谱和纲要.北京：科学出版社.2005
郭世绂.骨质疏松治疗的新进展.现代诊断与治疗,2003,14(5)：257～62
何召庆,秦泽荣,徐镔蕊,等.降钙素基因在乳酸杆菌中的克隆.中国兽药杂志,2002,36(6)： 20～22
黄建华,邱然,杨文洲,等.金属离子对啤酒酵母发酵中生成乳酸的影响.食品技术,2007(7)：123-124
黄丽金,陆兆新,袁勇军.响应面法优化德氏乳杆菌保加利亚亚种增殖培养基.食品科学,2005,26(5)：103～106
黄迎春,何壁梅,张义正,等.鲑鱼降钙素串联基因的克隆表达及重组蛋白的生物学活性.生物技术,2003,13(6)：8～10
姬华,乐国伟,王洪新,等.转基因食品的营养学评价研究进展.食品研究与开发,2009,30(7)：149～152
姜勇.鲑鱼降钙素在酵母中的多拷贝表达及安全性评价[博士学位论文].青岛：中国海洋大学,2008
姜勇,张学成,陈云松,等.rDNA介导的酵母表达鲑鱼降钙素基因多拷贝整合载体的构建.中国海洋大学学报,2009,39(3)：443～447
金汝城.均匀设计和正交设计在黄芪活性成份提取中的应用比较.生物数学学报,2009,24(3)：518-522
李国华,席小平,贾继峰,等.Wistar大鼠主要脏器系数与体重相关性的讨论.山西医药杂志,2005,34：661～662
李宏.微核试验的研究进展.安徽农业科学,2009,37(7)：2864～2866
李来好,郝志明,吴燕燕.基因食品的营养安全问题.现代食品科技,2005,21(2)：166～170
李宁.国内食品安全性毒理学评价的现状和发展.毒理学志,2007,21(5)：368～370
李宁.转基因食品的食用安全性评价.毒理学杂志,2005,6(19)：163～165
李寿祺,主编.卫生毒理学基本原理和方法.成都：四川科学技术出版社,1987,82
李兆鹏,张栩,徐斌.重组大肠杆菌高密度发酵中乳糖诱导表达hBLyS.过程工程学报,2005,5(4)：446～449
李岩,高虹,孙井江.Wistar大鼠体重和主要脏器参考值的研究.实用预防医学,2009,16(6)：1708～1711
李悠慧.我国食品毒理学发展50年.中国食品卫生杂志,1999,11(6)：5～6
李永仙,郑飞云,李崎.RSM法优化产p-葡聚糖酶的发酵培养基.食品工业科技,2009,30(8)：137-142
联合国粮农组织.食品安全风险分析(,樊永祥).北京：人民卫生出版社,2008
刘建中,熊亚红,翁丽萍,等.生物过程的优化.中山大学学报,2002,41：134～137
刘恺,张占吉,牛树启.转基因食品安全性评价综述.保定师范专科学校学报,2006,19(4)：18～20
刘深基,陈松森,张松,等.鲑鱼降钙素基因在大肠杆菌中的可溶性高效表达.基础医学与临床,1999,19(2)：89～93
刘向勇,张小华,鲍晓明.酒酵母工业菌株胁迫条件耐受性分析.中国酿造,2006(1)：8～11
刘颖,梁东春,郭刚,等.鲑鱼降钙素重组杆状病毒载体的构建及表达.现代口腔医学杂志,2003,17(1)：14～17
刘毓谷.卫生毒理学基础.北京：人民卫生出版社,1995
付立杰.现代毒理学及其应用.上海：上海科学技术出版社,2001
马复兴,邹复海.降钙素.中国医药学杂志,1983,3(3)：18～20
孟迅吾,周学瀛.骨质疏松症的诊断、预防和治疗.中华内分泌代谢杂志,1993,9(4)：246～248
潘良文.主要商业化转基因产品标准化检测技术的建立与应用[博士学位论文].南京：南京农业大学,2004
沈娟,陆兆新,别小妹,等.Bacillus sp.fmbJ224发酵产新型抗菌肽培养基的优化研究.生物工程学报,2005,4(21)：609～614
沈倩,胡德文,时春.神经网络应用技术.长沙：国防大学出版社,1993
史志诚.动物毒物学.北京：中国农业出版社,2001,6月,第一版
宋晓金.富含DHA的裂殖壶菌的工业化生产试验-脂肪酸提取及应用研究：[博士学位论文].青岛：中国海洋大学,2008
孙金旭,朱会霞.响应面法优化灰树花发酵培养条件研究.中国酿造,2009,9(210)：94～98
孙平楠.酵母表达鲑鱼降钙素基因载体的构建及相关研究[博士学位论文].青岛：中国海洋大学,2007
隋海霞,高艽,耿桂英,等.鹿茸人参制剂的毒理学安全性评价.现代预防医学,2007,34(23)：4448～4452
唐启义,冯明光.实用统计分析及其DPS数据处理系统.北京：科学出版社,2002
万琦,陆兆新.脱苦大豆多肽产生菌的筛选及其水解条件的优化.食品科学,2003,24(2)：29～32
王继花,薛永常,高圣.高渗胁迫下啤酒酵母的生理特性研究.中国酿造,2008,(45)：45～47
王普,孙立明,何军邀.响应面法优化热带假丝酵母104菌株产羰基还原酶发酵培养基.生物工程学报.2009,25(6)：863～868
王迁.欧美转基因食品法律管制制度比较研究.河北法学,2005,23(10)：114～121
王如平,马玉银.转基因产品的安全性评价.安徽农业科学,2008,36(36)：16097～16098,16139
王翔林,梁践,孙玉昆.降钙索基因工程研究.沈阳药学院学报,1995,12(4)：258～262
王晓辉,察少华,迟乃玉,等.海洋低温BS070623菌株选育及其发酵培养基优化(Ⅰ).渤海大学学报,2009,30(2)：97～100
王亚东,杨海燕,王海玉,等.SD大鼠30 d喂养试验正常参考值的探讨.现代预防医学,2007,34(7)：1265～1267
王忠华,周美园,夏英武.转基因产品的食用安全性评价.生命科学,2002,14(5)：305～307
文剑明,郑铭豪.破骨细胞的形成功能及细胞因子调节.中国骨质疏松杂志,1998,41：64～68
吴风霞,李纯厚,戴明.人工神经网络在海洋科学中的应用.南方水产,2009,5(1)：75～80
吴梧桐.生物制药工艺学.北京：中国医药科技出版社,1993.1-642
吴小平,孙洁胤.均匀设计在药学中的应用.浙江省医学科学院学报,2007,71：38～40
项华,章晓玲,查捷,等.SD大鼠生长发育指标和主要脏器正常参考值的探讨.中国卫生检验杂志,2008,18(2)：299～301
肖怀秋,李玉珍.微生物培养基优化方法研究进展.酿酒科技,2010(1)：90～94
谢恩C.加德.药物安全性评价.北京：化学工业出版社,2006：106～140
谢南.丙烯酰胺对雄性小鼠生殖系统毒性的研究[硕士学位论文].吉林：2007
杨冠珍,陈珍珍,崔大敷,等.人修饰型降钙素和鼠酰胺化酶基因在昆虫细胞中的偶连表达.生物工程学报,2002,18(1)：20～24
杨继良,项时康,杨伟华,等.棉子饼粉制取氨基酸水解液的研究.中国棉花,1995,22(4)： 8～10
杨永涛,涂国全.响应面法优化弗氏链霉菌S-221变种发酵培养基.生物加工过程,2005,3(2)：36～40
于峰,姚宝玉,王薏,等.小鼠畸形精子分型.上海实验动物科学,1997,17：232～233
余明泽,刘以农,蒋中仁等.环磷酰胺诱导小鼠骨髓细胞微核的影响因素的研究.现代预防医学,2007,34(5)：935～941
袁伯俊,廖明阳,李波.药物毒理学实验方法与技术.北京：化学工业出版社,2007.1～368
袁志发,周静芋.试验设计与分析(第二版).北京：高等教育出版社,2000.1～476
张虹,袁红梅.转基因食品安全性评价与管理.高师理科刊,2008,28(2)：71～74
张宏灿.豆饼酸水解制氨基酸复合液的研究.食品科学,1998,19(11)：59～61
张健,李夏明,汪仁官.两水平无重复析因设计的一个散度效应检验方法.工程数学学报,2006,23(4)：705～708
张桥,主编.卫生毒理学基础.北京：人民卫生出版社,2000.244～315
张军明.组织学与胚胎学实用彩色图谱.浙江：浙江大学出版社,2004.1～103
张希桥,杨志华.关于精子畸变分型的参考意见.卫生毒理学杂志.1995,9：46
张铣,刘毓谷.毒理学.北京：北京医科大学-中国协和医科大学联合出版社,1997.1～300
张玉彬,吴梧桐,吴文俊.降钙素的研究进展.中国医药学杂志,1996,31(8)：451～454
张绪金,唐英华.降钙素进展.中国骨质疏松杂志,2004,10(1)：96～97
曾北危.转基因生物安全.北京：化学工业出版社,2004.10～26
赵丽坤,郭会灿.微生物培养基优化方法概述,2008,20(4)：50～52
赵吉强,李霞,程智慧,等.鲑鱼降钙素基因向烟草遗传转化的初步研究.西北农林科技大学学报,2002,30：61～64
赵吉强,李霞,李丽霞,等.生菜遗传转化受体系统的建立及鲑鱼降钙素基因的导入.烟台大学大学学报(自然科学与工程版),2004,17(2)：116～121
郑定仙.我国食品毒理学回顾与展望.中国热带医学,2006,6(10)：1880～1881
郑莉,赵明,王会信.人降钙素基因在大肠杆菌中的克隆与表达.军事医学科学院院刊,1998,22(1)：23-26
中华人民共和国卫生部.GB15193.21-2003.食品安全性毒理学评价程序和方法.北京：中国标准出版社,2003
中华人民共和国卫生部.保健食品检验与评价规范(2003年版)(卫生部文件：卫法监发 (2003)42号).京,2003
中华人民共和国卫生部.食品安全性毒理学评价程序和方法(试行).北京：中国标准出版社,1985
中华人民共和国卫生部.GB15193-1-15193.1994.食品安全性毒理学评价程序和方法.北京：中国标准出版社,1995.56～57
中华人民共和国卫生部第28号令.转基因食品卫生管理办法,2001
中华人民共和国卫生部药政局.新药(西药)临床研究指导原则汇编(毒理学).1993：216～217
钟辉,彭英芳,李平,等.鲑鱼降钙素与骨生长肽融合基因的克隆及其在毕赤酵母中的表达.中国生物工程杂志,2002,22：79～82
钟为章,罗一菁,张忠智,等.红螺菌科光合细菌液体培养基的优化,2009,26(7)：67～69
周世祥,杨丽珠.试论转基因技术的安全性问题及其社会控制.成都理工大学学报(社会科学版),2006,14(1)：11～15
周一江,张学成,王玉梅,等.人/鲑降钙素嵌合基因在大肠杆菌中的串连表达.中国海洋大学学报,2007,37(1)：103～106,114
邹文安,刘宝,姜波等.基于Excel技术平台人工神经网络BP模型及应用.水文,2010,30(1)：44～46,63
GB 15193.5-2003,骨髓细胞微核试验[S].中国国家标准汇编(2003年修订-17.北京：中国标准出版社,2004,609～612
GB 15193.7-2003,小鼠精子畸形试验[S].中国国家标准汇编(2003年修订-17).北京：中国标准出版社,2004,619～622
Adow D A, Zwahlen C. Assessing environmental risks of transgenicplants. Ecology Letter,2006, 9:196～214
Anderson W A. The future relationship between the media,the food industry and the consumer. Br. Med. Bull.2000.56(1):254～268
Angela E, Kerry L.Dearfield, Michael C.Cimino. Mutagenicity test schemes and guidelines: U.S.EPA office of pollution prevention and toxics and office of pesticide programs. Environ Mole Mutagen,1993,21(1):38～45
Annadurai G. Design of optimum response surface experiments for adsorption of direct dye on chitosan. Biopronc Eng,2000,23:451～455
Arnold F H, Volkov A A. Directed evolution of biocatalysts. Curr Opin Chem Biol,1999,3: 54～59
Arntzen C J. Immunity for breakfast, Science,1998,280(8):831
Austin LA, Hunter H. Calcitonin:physiology and pathophysiology. N Engl J Med,1981,304(5): 269～276
Azria M, Copp D H, Zaneli J M.25 years of salmon calcitonin:from synthesis to therapeutic use. Calcif Tissue Int,1995,57:405
Barton J. Biotechnology for developing country agriculture-Intellectual property protection Washington, D.C:International Food Policy Research Institute,1999
Berg P, Singer M F. The recombinant DNA controversy:twenty years later. Bio/Technology. 1995,13,1132～4
Blanquet S, Marol-Bonnin S, Beyssac E, et al. The Biodrug concept:an innovative approach to therapy. Trends Biotechnol,2001,19:393～400
Blanquet S, Antonelli R, Laforet L, et al. Living recombinant Saccharomyces cerevisiae secreting proteins or peptides as a new drug delivery in the gut. Biotechnol,2004,110:37～49
Box GEP, Behnken DW. Some new three level designs for the study of quantitative variables. Technometrics.1960,2:455～475
Box GEP, Hunter, W.G, Hunter J S. Statistics for experiments:an introductory to design, data analysis, and model building. New Nork, John Wiley & Sons,1978
Bradbury A F, Finnie M D A, Smyth D G, et al. Mechanism of C-terminal amide formation by pituitary enzymes. Nature.1982,298:686～688
Chakraborty C, Nandi S, Sinha S. Overexpression, purification and characterization of recombinant salmon calcitonin, a therapeutic protein, in Streptomyces avermitilis. Protein Pept Lett,2004,11(2):165～173
Chambers, T. J., and Magnus, C. J. Calcitonin alters behaviour of isolated osteoclasts. J Pathol, 1982,136:97～106
Chi Z, Liu J, Zhang W. Trehalose accumulation from soluble strarch by Saccharomycopsis fibuligera sdu. Enzyme Microb TechnoL,2001,28:240～245
Chowdhury E H, Kuribara H, Sultana P. Detection of corn intrinsic and recombinant DNA fragment and Cryl Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt. Journal of animal science,2003,81:2546～2551
Ceserani R. Calcitonin and prostaglandin System. Life Sci,1979,25:1851～1856
Cockburn A. Assuring the safety of genetically modified (GM) foods:the importance of a holistic. Integrative approach Biotechnology,2003,98(1):79～106
Codex Alimentarius Commission. Joint FAO/WHO food standard programme. Chiba, Japan,2000: 14～17
Copp, D.H., Cameron, E. C., Cheney, B. A., Davidson, A. G. F., and Heinze K. G. Evidence for calcitonin-A new hormone from the parathyroid that lowers blood calcium. Endocrinology.1962 a 70:638～649
Copp, D. H. and Cheney, B. A. Calcitonin-A hormone from the parathyroid that lowers the calcium level of the blood. Nature.1962 b,193:381～382
Copp, D. H., Niall, H. D., Keutmann, H. T. and Potts J. T. Amino acid sequence of salmon ultimobranchial calcitonin. Proc Natl Acad Sci USA.1969,64:771～778
Cott MK, Vining GG, Douglas CM, et al. Modifying a central composite design to model the process mean and variance when there are hard-to-change factors. App.l Statist.2006,55: 615～630
Cranney A, Tugwell P, Zytaruk N, et al. Meta2Analysis of Calcitonin for the Treatment of Postmenopausal Osteoporosis. Endocrine Reviews,2002,23(4):540～51
Dan YE, Zhi-nan Xu and Pei-lin CEN. Medium Optimization for Enhanced Production of Cytosine-substituted Mildiomycin Analogue (MIL-C) by Streptoverticillium rimofaciens ZJU 5119. J Zhe-jiang Univ Sci B,2008,9(1):1581～1673
Daniel Heller. A Review and Applications of Flow Cytometry.2004,1-13
Day A, Meyling J C. Practical experience with rapid methods of detecting yeasts in breweries. J Inst Brew,1990,89:204～206
Desai K M, Akolkar S K, Bae Y P, et al. Optimization of fermentation media for exopoly saccharide production from Lactobocillue plantarum using artificial intelligence-based tech-niques. ProcessBiochemistry,2006,41:1842～184
Eipper B A, Perkins S N, Husten E J, et al. Peptidyl-α-hydroxyglycine|α-amidatinglyase. J Biol Chem,1991,266:7827-7833
Ellstand N C, Prentice H C, Hancock J F. Gene flow and introgression from domesticated plants into their wild relatives. Annual Review Of Ecology and Systematics,1999,30:539～563
Ellstand N C. Current knowledge of gene flow in plants:Implications for transgene flow. Philosophical Transactions of the Royal Society B. Biological Sciences,2003,358:1163-1170
Ertola R J, Giulietti AM, Castillo F J. Design, Formulation and Optimization of Media. Bioprocess Technol,1995,21:89～137
Ewen S W, Pusztai A. Health risks of genetically modified food. Lancet,1999,354:684～690
EU. Commission of the European Communities. Communication from the commission on the precautionary principle COM 1. Brussels.2000
Fang KT, Winker P. Uniform design. Theory and Applications. Technometrics.2000,42:237～248 FAO/WHO. Biotechnology and food safety. RomeL Report of Second joint FAO/WHO Consulta-tion,1996
FAO/WHO. Evaluation of allergenicity of genetically modifiedfood. Report of a joint FAO/WHO expert consultation on allergenicity of food derived from biotechnology. FAO, Rome,2001
FAO/WHO. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechno-logy, Safety Aspects of Genetically Modified Foods of Plant Origin. World Health Organisation, Publication details,2000
Fenech M. The cytokinesis-block micronucleus technique:A detailed description of the method and its application to genotoxicity studies in human populations. Mutation Res,1993,285:35～44
Feng Y, He Z, Ong SL. Optimization of agitation, aeration, and temperature conditions for maximum β-mannanse production. Enzyme Microb TechnoL,2003,32:282～289
Foster G V, Baghdiantz A, Kumar M A, Slack E, Soliman H A and MacIntyre I. Thyroid origin of calcitonin. Nature,1964,202:1303～1305
Foster GV, MacIntyre I, and Pearse AGE. Calcitonin production and the mitochondrion-rich cells of the dog thyroid. Nature,1964,203:1029～1030
Foy CA, Parkes HC. Emerging homogeneous DNA-based technologies in the clinical laboratory. Clin Chem,2001,47(6):990～1000
Franco-Lara E, Link H, Weuster-Botz D. Evaluation of artificial neural networks for modeling and optimization of medium composition with a genetic algorithm. Process Biochemistry,2006,41: 2200～2206
Gennari C. Dolore osseo. Endorfine calcitonine. In:Gennari C, Segre G. eds. The effects of calcitonin in man. Proc.lst int. Wkshp. Florence 1982. Masson, Milan1983.213～222
George A S. Challenges for methods to detect genetically modified DNA in foods. Food Control, 1999,10:351～352
Grillot-courvalin C, Goussard S, Huetz F, et al. Functional gene transfer from intracellular bacteria to mammalian cells. Nature Biotechnol,1998,16:1～5
Hai-feng Pan, Wen-na Bao, Zhi-peng Xie. Optimization of medium composition for cisepoxysuccinate hydrolase production in Escherichia coli by response surface methodology. African Journal of Biotechnology,2010,9(9):1366～1373
Heddle J A. A rapid in vivo test for chromosomal damage. Mutal Res,1973,18:187～190
Henriksen DB, Rolland M, Jakobsen MH, et al. C-terminal amidation of calcitonin by carboxype-ptidase Y catalyzed transpeptidation with a photocleavable nucleophile. Pep Res,1992,5(6):321-342
Hiatt A, Cafferkey R, Bowdish B. Production of antibodies in transgenetic plants. Nature,1989, 342:76～78
Hirsch PF, Gauthier GF, Munson PL. Thyroid hypocalcaemic principle and recurrent laryngeal nerve injury as factors affecting the response to parathyroidectomy in rats. Endocrinology,1963, 73:244～250
Hohmann S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev, 2002,66(2):300～372
Huppatz F L. Fitzgerald P A. Genetically-modified foods-safety and regulatory issues. J. Aust. 2000,172(4):170～173
Isar J, Agarwal L, Saran S, et al. A statistical approach to study the interactive effects of process parameters on succinic acid production from Bacteroidesfragilis. Anaerobe,2007,13:50～56
Ishikawa J, Kawaguchi J, Tamaoki H. Production of human calcitonin as tandemly repeat fusion protein. JP05320196,1993-13-3
Kishor CU, Kamlesh CP. Statistical screening ofmedium components by Plackett-Burman design for lactic acid production byLactobacillussp. KCP 01 using date juice. Bioresource Technology, 2007,98:98～103
Klotz A, Mayer J, Einspanier R. Degradation and possible carry over of feed DNA monitiored in pigs and poultry. Food Rearch Technology,2002,214:271～275
Koji Satake, Toshiaki Monaka, Satoshi Yamada et al. The rapid development of diesel engines using an optimization of the fuel injection Control. Mitsubishi Heavy Industries,2008,45:6-10
Kowalchuk G A, Bruinsma M, Van Veen J A. Assessing responses of soil microorganisms to GM plants. Trends in Ecology and Evolution,2003,18:403～410
Kuiper H A, Kleter G A, Kok E J.Assessment of the food safety issues related to genetically modified food. Plant,2001,27(6):503～528
Kumar MA, Foster GV, MacIntyre I. Further evidence for calcitonin:A rapid acting hormone which lowers plasma calcium. Lancet ii,1963:480～482
Leeder S R. Genetically modified foods-food for thought. Med. J. Aust.2000,172(4):173～174
Lems WF, Dijkmans BA. Effect van anti-osteoporotic medicine on het on tstaan van wervelfracyuren. Ned Tijdschr Geneeskd,2000,144:1941～1945
Liu C, Ruan H, Shen H, et al. Optimization of the Fermentation Medium for Alpha-galactosidase Production from Aspergillus Foetidus ZU-G1 Using Response Surface Methodology. J Food Sci, 2007,72(4):120～125
Losey J E. Transgenic pollen harms monarch larvae. Nature,1999, (399):214
Lu BR, Snow AA. Gene flow from genetically modified rice and its environmental consequences. Bioscience,2005,55:669～678
Rajasimman M, Subathra S. Optimization of Gentamicin Production:Comparison of ANN and RSM Techniques. International Journal of Chemical and Biomolecular Engineering,2009,14～19
MacIntyre I, Parsons J A, Robinson C J. The effect of thyrocalcitonin on blood bone calcium equilibrium in the perfused tibiae of the cat. J Physiol,1967,191:393～405
Mahony J, Chong S, Jang D, et al. Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chamydia trachomatis nucleic acid by PCR, ligase chain reation, and transcription-mediated amplification:identification of urinary substances associated with inhibition and removal of inhibitory activity. J Clin Microbiol,1998,36:3122～3126
Marrazzo JM, Handsfield HH, Whittington WL. Predicting chlamydial and gonococcal cervical infection:implications for management of cervicits. Obstet Gynecol,2002,100(3):579～584
Marrs B, Delagrave S, Murphy D. Novel approaches for discovering industrial enzymes. Curr Opin Microbiol,1999,2:241～245
Mason HS, Ball J M, Shij j. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immuno genicity in mice. Pro Natl Acad Sci USA,1996,93:5335～5340
Metcalfe D D, Astwood J D, Townsend R, et al. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit Rew Food Sci Nutr,1996,36(S):165～186
Millstone E, Brunner E, Mayer S. Beyond"substantial equivalence". Nature,1999,401:525～526
Mironova R, Philipova D, Marekov L, et al. Secretion of oligomeric Val8-human calcitonin by Saccharomyces cerevisiae. FEMS Microbiol Lett,1991,83:23～28
Mosely B E. Safety assessing and public concern for genetically modified food Products:The European view. Toxicol Pathol,2002,30(1):129～131
Muralidhar R V, Chirumamila R R, Marchant R, et al. A response surface approach for the comparison of lipase production by Candida cylindracea using two different carbon source. Biochem Eng J,2001,9:17～23
Neher R, Riniker B, Maier R, Byfield PGH, Gudmundsson TV, MacIntyre I. Human calcitonin. Nature,1968,220:984～986
Nuti R, Vatimmo A, Martini G, et al. Carbocalcitonin treatmenl in Sudeck's atrophy. Clin Orthop Rel Res,1987,215:217-222
OECD. Food Safety Evaluation. Paris:organization for Economic Cooperation and Development. 1996
OECD. Safety evaluation of foods produced by modern biotechnology:concerps and principles. Paris:DECD,1993:1～16
OECD. Report of the OECD workshop on the toxicological and nutritional testing of novel foods, Aussois, France,5-8 March 1997. Paris:organization for Economic Cooperation and Developme-nt.1998
OECD Consensus Doeuments. Paris:orgnisation for Economic Cooperation and Development, Inter-Agency Network for Safety in Biotechnology.2001 a
OECD. Report of the OECD workshop on Nutritional Assessment of Novel Foods and Feeds, Ottawa, Canada,5-7 February 2000. Paris:orgnisation for Economic Cooperation And Development.2001 b
Ofoghil H, Moazami] N, Domonsky N N, et al. Cloning and expression of human calcitonin genes in transgenic potato plants. Biotechnology Letters,2000,22:611～615
Parsell A D, Sauer R T. The structural stability of a protein is an important determinant in Escherichia coli. Biol Chemistry,1989,264:7590～7596
Potts J T, Niall H D, Keutmann H T, Brewer, H B, Deftos L J. The amino acid sequence of porcine thyrocalcitonin. Proc Natl Acad Sci,1968,59:1321～1328
Ray M V, Duyne P V, Bertelsen A H, et al. Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide. Biotechnology (NY),1993, 11(1):64～70
Ray M V, Meenan C P, Consalvo A P, et al. Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli. Protein Expr Purif,2002,2S(2):249～259
Ray M V, Van Duyne P, Bertelsen A H, et al. Production of recombinant salmon calcitonin by in vit ro amidation of an E. coli. produced precursor peptide. Biotechnology,1993,11(1):64～70
Reddy PRM, Reddy G, Seenayya G. Production of thermostable pullulanase by Clostridium the rmosulfuro genes SV2 in solid-state fermentation:optimization of nutrients levels using response surface methodology. Bioproc Eng,1999,21:497～503.
Reginster JY. Treatment of bone in elderly subjects:calcium, vitamin D, fluor, bisphosphonates, calcitonin. Horm Res,1995,43:83～88
Rittel W, Brugger M, Kamber B, Riniker B, Sieber P. Thyrocalcitonin.Ⅲ. Die Syntheses des alpha-thyrocalcitonins. Helv Chim Acta,1968,51:924～928
Roukas T, LiakopouLou-Kyriakides M. Production of pullulan from beet molasses by Aureobasi-dium pullulans in a stirred tank fermentor. J Food Eng,1999,40:89～94
Ruth E. Montes, Grady Hanrahan, Frank A. Gomez. Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary Electrophoresis, 2008,29:3325～3332
Sato N, Mizumoto K, Makamura M, et al. A possible role for centrosome overduplication in radiation induced cell death. Oncogene,2000,19:5281～5290
Saudagar P S. Singhal R S. Optimization of nutritional requirements and feeding strategies for clavulanic acid production by streptomyces clavuligerus. Bioresour Technol,2007,98(10):2010-2017
Shadrack R morphuti. Cartagena protocol on biosafety to the convention biological diversity. Registrar:GMO,2000
Simth N. Seeds of opportunity and assessment of the benefits safety and oversight of plant genomics and agricultural biotechnology. Committee Review of Entomology,1996, (11): 106～111
Sofuni T. Japanese guidelines for mutagenicity testing. Environ Mole Mutagen,1993,21(1):2-7
Stewart C N, Halfhill M D, Warwick S I. Transgene introgression from genetically modified crops to their wild relative. Nature Reviews of Genetics,2003,4:806～817
Sun Ping-nan, Zhang Xue-cheng, Zang Xiao-nan, et al. Anti-hypercalcemic effect of orally adm-inistered recombinant Saccharomyces cerevisiae expressing salmon calcitonin on hypercalcemic rats. Biotechnol Lett.2007,29(7):1013-1018
Tacket CO, Mason HC, Losonsky G, et al. Immunogenicity in human so far recombinant bacterial antigen deliver edina transgenic potato. Nature Medicine,1998, (14)5:607～609
Thompson D R. Response surface experimentation. J Food Proc Pres,1982, (6):155～188
Tushar Gulati, Mainak Chakrabarti, Anshu Singh, et al. RSM and ANN/GA for Optimization of Soybean. Food Technol. Biotechnol.2010,48(1):11～18
Tuysuz B, Mercimek S, Unger S, et al. Calcitonin treatment in osteoectasia with hyper-phosphas-i (juvenile Paget's diaease):Radiographic change after treatment. Pediatr Radiol,1999,29: 838～841
Wang F Q, Gao C J, Yang C Y. Optimization of all Ethanol Production Medium in very High Gravity Fermentation. Biotechnol Lett,2007,29(2):233～236
Watkinson A R, Freckleton R P, Robinson R A, et al. Predictions of biodiversity response to gene-tically modified herbicide-tolerant crops. Science,2000,289:1554～1557
Waynforth B.H. Injection techniques experimental and surgical techniques in the rat. London: Academic Press,1980.3～61
Wyrobek A J, Bruce W R. Chemical induction of sperm abnormalities in mice. Proc. Nat. Acad. Sci. USA,1975,72(11):4425～4429
Xu CP, Yun JW. Optimization of Submerged-culture Sonditions for Mycelial Growth and Exobiopolymer Production by Auricularia Polytricha (wood ears fungus) using the Methods of Uniform Design and Regression Analysis. Biotechnol Appl Biochem,2003,38:193～199
Yatuba M, Suzuki Y, Ohsuye K, et al. High expression of a recombinant human calcitonin precu-rsor peptide in Escherichia coli. Appl Microbiol Biotechnol,1995,42(5):703～708
Zaidi M, Fuller K, Bevis PJR, Gaines Das RE, Chambers TJ, MacIntyre Ⅰ. Calcitonin gene-related peptide inhibits osteoclastic bone resorption:A comparative study. Calcif Tissue Int,1987,40: 149～154