重组大肠杆菌DH5α生产rNGR-Tum-5工艺的研究
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摘要
本文包括五个章节。从发酵培养基,摇瓶培养条件,5L发酵罐培养条件三个方面对重组大肠杆菌DH5α生产rNGR-Tum-5的能力进行研究。使得在高密度培养情况下工程菌细胞终浓度OD_(600)达到16.0,rNGR-Tum-5表达量达到30%,rNGR-Tum-5的生产能力达到4.80。
1.重组大肠杆菌DH5α发酵培养基的筛选
在摇瓶中对大肠杆菌DH5α的发酵培养基进行研究,首先从几种常用的培养基中筛选出LB培养基作为基础培养基。在此基础上,对培养基中的碳源、氮源、微量元素进行筛选,得出在培养基组分(%)为:甘油1,酵母粉3,蛋白胨1,氯化钠0.5,微量元素母液0.15时,重组大肠杆菌DH5α生产rNGR-Tum-5的能力最强,较初始培养基提高了3倍。
2.重组大肠杆菌DH5a摇瓶发酵的筛选
在摇瓶中,研究发酵工艺条件如温度、溶氧量、种子菌龄、诱导时机、诱导剂浓度、诱导表达时间等对工程菌生长情况和蛋白表达的影响。得出最佳培养条件为:培养温度37℃,摇床转速200rpm,种子菌龄选择在OD_(600)为1.0,在对数生长中期(OD_(600)=2.0,4h处)时诱导,诱导剂浓度选择在0.5 mmol/L,诱导表达时间为4h。
3.重组大肠杆菌DH5α在5L发酵罐中的培养
根据工程菌高密度发酵的生长曲线,确定诱导时机。优选出合适的接种量8%,得出发酵罐中工程菌生长和蛋白表达情况。控制pH时采用二阶段培养法,前期菌体生长和后期蛋白表达维持在不同的pH范围;使工程菌生产rNGR-Tum-5的能力有所提高。对发酵产物进行分离纯化鉴定,在分子量约10~4Da处出现一条蛋白带,为目标蛋白。
The desertive consists of five chapters: The productivity of rNGR-Tum-5 from recombinant E.coli DH5αwas systematically studied, which includes optimization of fermentation culture medium, culture conditions in both shake flask and 5L fermenter, respectively. After high-density culture, the final cell growth of engineering bacteria OD_(600) reached 16.0, the expression of rNGR-Tum-5 reached 30%, and the productivity of rNGR-Tum-5 from recombinant E.coli DH5αreached 4.80.
1. Optimization of the culture media of recombinant E.coli DH5α
To improve the yield of rNGR-Tum-5 produced from recombinant E.coli, LB culture medium was chosen as the basic medium from several types of common medium. The influences of different types of carbon, nitrogen sources and trace elements in the culture medium on the yield of rNGR-Tum-5 had been investigated with shake flask method. It was found that the yield of rNGR-Tum-5 was the highest and it raised five times over that in the initial culture medium under the condition of 1% glycerol, 3% yeast extract, 1% peptone, 0.5% sodium chloride and 0.15% trace elements solution in the culture medium (w/w).
2. Culture of recombinant E.coli DH5a in shake flask
The influences of fermentation conditions such as temperature, dissolved oxygen, inoculum age, induction time, inducer concentration, expression time on the growth of bacteria and protein expression has been studied. The best culture conditions were concluded as the following: culture temperature 37℃, shaking speed 200 rpm, inoculum age OD_(600) 1.0, induction at mid-logarithmic growth (OD_(600)= 2.0, 4 h ), IPTG concentration chosen at 0.5mmol/ L, expression time for 4h.
3. 5L fermentor culture of recombinant E.coli DH5α
Initially the appropriate seed amount of 8% was confirmed. The engineering bacterial growth and protein expression in fermentor were obtained. The ability of engineering bacterial producing rNGR-Tum-5 has been raised by controlling pH, which adoptted two-stage culture method. The pH in the early stage of cell growth was different from that in the late stage of protein expression, which raised significantly the yield of rNGR-Tum-5 . The product of fermentation has been separated and determined. The target protein appeared about 10~4 Da in the molecular weight.
1.重组大肠杆菌DH5α发酵培养基的筛选
在摇瓶中对大肠杆菌DH5α的发酵培养基进行研究,首先从几种常用的培养基中筛选出LB培养基作为基础培养基。在此基础上,对培养基中的碳源、氮源、微量元素进行筛选,得出在培养基组分(%)为:甘油1,酵母粉3,蛋白胨1,氯化钠0.5,微量元素母液0.15时,重组大肠杆菌DH5α生产rNGR-Tum-5的能力最强,较初始培养基提高了3倍。
2.重组大肠杆菌DH5a摇瓶发酵的筛选
在摇瓶中,研究发酵工艺条件如温度、溶氧量、种子菌龄、诱导时机、诱导剂浓度、诱导表达时间等对工程菌生长情况和蛋白表达的影响。得出最佳培养条件为:培养温度37℃,摇床转速200rpm,种子菌龄选择在OD_(600)为1.0,在对数生长中期(OD_(600)=2.0,4h处)时诱导,诱导剂浓度选择在0.5 mmol/L,诱导表达时间为4h。
3.重组大肠杆菌DH5α在5L发酵罐中的培养
根据工程菌高密度发酵的生长曲线,确定诱导时机。优选出合适的接种量8%,得出发酵罐中工程菌生长和蛋白表达情况。控制pH时采用二阶段培养法,前期菌体生长和后期蛋白表达维持在不同的pH范围;使工程菌生产rNGR-Tum-5的能力有所提高。对发酵产物进行分离纯化鉴定,在分子量约10~4Da处出现一条蛋白带,为目标蛋白。
The desertive consists of five chapters: The productivity of rNGR-Tum-5 from recombinant E.coli DH5αwas systematically studied, which includes optimization of fermentation culture medium, culture conditions in both shake flask and 5L fermenter, respectively. After high-density culture, the final cell growth of engineering bacteria OD_(600) reached 16.0, the expression of rNGR-Tum-5 reached 30%, and the productivity of rNGR-Tum-5 from recombinant E.coli DH5αreached 4.80.
1. Optimization of the culture media of recombinant E.coli DH5α
To improve the yield of rNGR-Tum-5 produced from recombinant E.coli, LB culture medium was chosen as the basic medium from several types of common medium. The influences of different types of carbon, nitrogen sources and trace elements in the culture medium on the yield of rNGR-Tum-5 had been investigated with shake flask method. It was found that the yield of rNGR-Tum-5 was the highest and it raised five times over that in the initial culture medium under the condition of 1% glycerol, 3% yeast extract, 1% peptone, 0.5% sodium chloride and 0.15% trace elements solution in the culture medium (w/w).
2. Culture of recombinant E.coli DH5a in shake flask
The influences of fermentation conditions such as temperature, dissolved oxygen, inoculum age, induction time, inducer concentration, expression time on the growth of bacteria and protein expression has been studied. The best culture conditions were concluded as the following: culture temperature 37℃, shaking speed 200 rpm, inoculum age OD_(600) 1.0, induction at mid-logarithmic growth (OD_(600)= 2.0, 4 h ), IPTG concentration chosen at 0.5mmol/ L, expression time for 4h.
3. 5L fermentor culture of recombinant E.coli DH5α
Initially the appropriate seed amount of 8% was confirmed. The engineering bacterial growth and protein expression in fermentor were obtained. The ability of engineering bacterial producing rNGR-Tum-5 has been raised by controlling pH, which adoptted two-stage culture method. The pH in the early stage of cell growth was different from that in the late stage of protein expression, which raised significantly the yield of rNGR-Tum-5 . The product of fermentation has been separated and determined. The target protein appeared about 10~4 Da in the molecular weight.
引文
[1]卢起,涂文坚.生物制药的现状与发展[J].中外健康文摘,2008,5(6):186-188
[2]何忠平.世界制药工业发展及应对[J].医药快讯信息,2004,10(2):18-22
[3]颜真,张英起.蛋白质研究技术[M].西安:第四军医大学出版社,2007:1-2
[4]陈贯虹,迟建国,邱维忠.多肽药物的研究进展[J].山东科学,2008,21(3):42-46
[5]马楠,张英起,颜真.重组人Tum-5融合蛋白的克隆、表达、纯化及鉴定[J].第四军医大学学报,2005,26(14):1279-1281
[6]侯登勇,张英起,韩苇等.TRAIL与导向肽融合基因的克隆、表达及其表达产物的生物学活性[J].中国生物制品学杂志,2005,18(4):277-279
[7]王慧,颜真,赵宁等.RGD特异性结合多肽与重组改构人肿瘤坏死因子融合基因的克隆表达及其抑瘤活性[J].中国生物制品学杂志,2005,18(3):185-189
[8]Jieru Meng,Nan Ma,Zhen Yan,et al.NGR Enhanced the Anti-Angiogenic Activity of tum-5[J].Biochem,2006(140):1-6
[9]马楠.靶向性重组人Tum-5融合蛋白的克隆、表达、纯化及生物学活性的测定[D].西安:第四军医大学硕士学位论文,1995
[10]Folkman J..What is the evidence that tumors are angiogenesis dependent?[J].Natl Cancer Inst,1990,82(1):4-6
[11]Hanahan D.,Forlman J..Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis[J].Cell,1996,86(3):353-364
[12]Petitclerc E.,Boutaud A.,Prestayko A.,et al.New function for non-collagen type Ⅳ.Novel integrin ligands inhibting angiogenesis and tumor growth in vivo[J].Biol Chem,2000,275(11):8051-8061
[13]Maeshima Y.,Colorado PC,Torre A.,et al.Distint antitumor propertiesof a type Ⅳcollagen domain derived from basement membrane[J].Biol Chem,2000,275(28):21340-21348
[14]Maeshima Y.,Sudhakar A.,Lively JC,et al.Tumstatin,an endothelial cell-specific inhibitor of protein synthesis[J].Science,2002,295(5552):140-143
[15]Maeshima Y.,Yerramalla UL.,Dhanabal M.,et al.Extracellular matrix-derived peptide binds to alpha v beta 3 integrin and inhibits angiogenesis[J].Biol Chem,2001,276(34):31959-31968
[16]Kashtan CE.Alport syndrome and thin glomerular basement membrane disease[J].Am Soc Nephrol,1998,9(9):1736-1750
[17]Sudhakar A.,Sugimoto H.,Yang C.,et al.Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha Ⅴ beta 3 and alpha 5 beta integrins[J].Proc Natl Acad Sci USA,2003,100(8):4766-4771
[18]Maeshima Y.,Colorado PC,Kalluri R..Two RGD-independent alpha ⅴ beta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties[J].Biol Chem,2000,275(31):23745-23750
[19]顾取良,罗进贤,张添元等.人tumstatin基因的克隆、表达和抗体制备[J].中山大学学报,2005,44(2):189-192
[20]黄敏丽,罗国容,陈维平.Tumstatin肽对视网膜微血管内皮细胞增生和迁移的影响[J].眼科新进展,2008,28(9):649-652
[21]高云,俞悦,陆森.人肿瘤抑素tumstatin的基因克隆及其原核表达[J].中国生化药物杂志,2005,26(6):324-327
[22]Hamano Y.,Kalluri R..Tumstatin,the NC1 domain of a3chain of type Ⅳcollagen,is an endogenous inhibitor of pathological angiogenesis and sup resses tum or growth[J].Biochem Biophys Res Commun,2005,333(2):292-298
[23]Maeshima Y.,Manfredi M.,Reimer C.,et al.Identification of the anti-angiogenic site with in vascular basement membrane-derived tumstatin[J].Biol Chem,2001,276(18):15240-15248
[24]Netzer KO,Leinonen A.,Boutaud A..The goodpasture autoantigen.Mapping the major conformational epitope(s) of alpha3(Ⅳ) collagen to residues 17-31 and 127-141 of the NC1 domain[J].Biol Chem,1999,274(16):11267-11274
[25]Hellmark T.,Burkhardt H.,Wieslander J..Goodpasture disease.Characterization of a single conformational epitope as the target of pathogenic autoantibodies[J].Biol Chem,274(36):25862-25868
[26]任娜,王梁华,高云等.Tumstatin_(183-230)-TRAIL融合蛋白的克隆表达及其生物学双功能鉴定[J].第二军医大学学报,2008,29(5):474-478
[27]Qian Xiaohong,Xie Jianwei.The application of modern instrument analysis to the study of biomedicine[M].Beijing,Chemical Industry Press,2003:21-22
[28]Holzgrabe U.,Wawer I.,Diehl B..NMR spect roscopy in drug development and analysis [M].New York,1999:23-24
[29]Arap W.,Pasqulini R.,Ruoslahti E..Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model[J].Science,1998,279:377-380
[30]Folkman J..Angiogenesis in cancer,vascular,rheumatoid and other disease[J].Nat Med,1995,1(1):27-31
[31]Pasqualini R..Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis[J].Cancer Res,1997,15(6):722-727
[32]Curnis F.,Angelina S.,Laura B.,et al.Enhancement of tumor necrosis factor α antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N(CD13)[J].Nature Biotechnol,2000,18(2):1185-1190
[33]李民,陈常庆.重组大肠杆菌高密度发酵研究进展[J]_生物工程进展,2000,20(2):26-31
[34]孙柏欣,刘长远,陈彦.基因表达系统研究进展[J].现代农业科技,2008,2:205-208
[35]刘爽.原核系统可溶性表达策略[J].生物技术通讯,2005,16(2):172-175
[36]郭井利,彭永刚,刘鹏.重组蛋白在大肠杆菌包含体蛋白的表达及其复性[J].畜牧兽医科技信息,2005(8):65-67
[37]谢磊,孙建波,张世清.大肠杆菌表达系统及其研究进展[J].华南热带农业大学学报,2004,10(2):16-19
[38]沈毅,姚见儿,易进华等.重组大肠杆菌高密度培养的进展[J].药物生物技术,2000,7(4):243-247
[39]吴丹,仇华吉,童光志.几种表达系统的比较[J].生物技术通报,2002,2:30-34
[40]马莉.人粒细胞.巨嗜细胞集落刺激因子基因克隆,表达,摇瓶培养及分离纯化的研究[D].西安:西北大学学位论文,2004
[41]戎晶晶,刁振宇,周国华.大肠杆菌表达系统的研究进[J].药物生物技术,2005,12(6):416-420
[42]郭广君,吕素芳,王荣富.外源基因表达系统的研究进展[J].科学技术与工程,2005,6(5):582-586
[43]罗红霞,林少华,任发政.重组大肠杆菌BL21(DE3)/rbLF-N高密度培养条件优化[J].中国乳品工业,2007,35(11):10-15
[44]方方,沈佳胤,殷志敏等.乳糖诱导重组人TRA IL胞外片段在大肠杆菌中的表达[J].安徽农业大学学报,2008,35(2):266-270
[45]熊宗贵.生物技术制药[M].北京:高等教育出版社,2000,76-77
[46]DaeHyuk Kweon,Nam Soo Han,Moon Park,et al.Over 2 production of Phytolacca insularis protein in batch and fed-batch of recombinant Escherichia coli Process[J]. Biochemistry,2001,36:537-542
[47]陈航,黄晓南,陈春等.重组SOD的摇瓶发酵条件研究[J].医学理论与实践,2006,19(11):1255-1257
[48]孟文丽,许云禄.蛇毒蛋白基因的大肠杆菌表达研究进展[J].药学专论,2008,17(15):3-5
[49]吴一凡,张双全,高秀玉等.乳糖诱导pET载体表达重组蛋白的研究[J].南京师大学报,2002,25(1):89-93
[50]褚仲梅,申秀云,刘现杰等.以乳糖为诱导剂的高密度发酵[J].中国医药工业杂志,2001,32(11):491-494
[51]Betenbaugh MJ.Biotechnol[J].Bioeng,1989,33:1425-1436
[52]王瑞霞,张兆红,刘玉霞等.枯草芽孢杆菌B-903最佳发酵时间研究[J].河南农业科学,2005,3:46-48
[53]郑杭民.生物工程进展[M].北京:科学技术文献出版社,1990:135-136
[54]杨炜,王伟刚,田海英等.重组大肠杆菌高表达高密度发酵研究[J].生物技术,2006,16(3):83-86
[55]陈蔚青,陈虹,胡文浪等.重组人胰岛素样生长因子-Ⅰ工程菌高密度发酵工艺优化[J].中国生化药物杂志,2008,29(2):121-124
[56]欧俊杰.重组人干细胞因子的生产工艺研究[D].西安:西北大学学位论文,2004
[57]Riesenberg D.High-cell-density cultivation of Escherichia coli[J].Current Opinion in Biotechnology,1991,2(3):380-384
[58]Markl H.,Zenneck C.,Dubach A,et al.Cultivation or E.coli to high cell densities in a dialysis reactor[J].Microbiotechnol,1993,39(5):48-52
[59]LeeSY.High cell density culture Escherichia coli[J].TrendsBiotechnol,1996,14(3):98-105
[60]方宏清.生物工程进展[M].北京:科学出版社,1992
[61]MoriH..ChemEng[M].JPn,1979:211-214
[62]Shimizu N..FermentTeehno[M].JPn,1988:159-160
[63]倪雪朋,熊三玉,管斌.利用流加发酵进行耐氧两歧双歧杆菌高密度培养的研究[J].中国食品添加剂,2007,11(2):114-117
[64]Reisenberg D.APPI.Mierobiol[J].Bioteehol,1990,34(2):77-82
[65]张泉,于可响,郭晓宇等.重组大肠杆菌的高密度发酵放大工艺研究[J].扬州大学学报,2007,28(1):17-20
[66]乔世伟.微生物连续发酵过程的有关计算[J].中国调味品,2002,11(11):3-8
[67]王江龙,孜力汗,白凤武.絮凝酵母SPSC01连续培养最适生长条件的研究[J].微生物学报,2004,44(1):93-95
[68]刘如林.微生物工程概论[M].天津:南开大学出版社,1995:53
[69]沈萍.微生物学[M].北京:高等教育出版社,2000:135
[70]陈坚,堵国成,李寅等.发酵工程实验技术[M].北京:化学工业出版社,2003:101-102
[71]丛春水,苏志国,邓继先.恒pH葡萄糖流加方法培养重组大肠杆菌生产人肿瘤坏死因子-α及动力学研究[J].微生物学杂志,1998,18(4):44-47
[72]顾维新.重组大肠杆菌的发酵与外源基因的表达[J].海峡药学,2007,19(2):84-87
[73]艾恒.包涵体研究进展[J].中国医学文摘,2006,27(2):109-111
[74]M J Weickert,D H Doherty,E A Best,et al.Optimization of heterologous protein production in Escherichia coli[J].Currop in Biotechnol,1996,7:494-499
[75]丁兰宝,牛丹丹,石贵阳.大肠杆菌表达古菌基因的发酵条件研究[J].研究与探讨,2008,29(2):97-100
[76]霍向东,石玉瑚.大肠杆菌高密度发酵补料调控策略的研究进展[J].新疆农业科学,2004,41:16-18
[77]温进坤,韩梅.医学分子生物学[M].北京:科学出版社,1999,209-210
[78]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000,89-90
[79]D.R.马歇克,J.T.门永,R.R.布格斯.蛋白质纯化与鉴定实验指南[M].北京:科学出版社,2000:72-73
[80]骆红琴,李乃,孙文晓.一种新型金属螯合亲和色谱固定相的制备及评价[J].天津理工大学学报,2007,23(2):59-62.
[81]李蓉,陈国亮,赵文明.固定金属离子亲和色谱-蛋白质分离的方法、原理、特性和应用[J].化学通报,2005,5:352-360.
[82]Ueda E KM,Gout PW,MorgantiL[J].Chromatogr.A,2003,988:1-23.
[83]杨利,贾凌云,邹汉法.IDA型固定化镍离子金属螯合亲和膜色谱对人血清白蛋白的分离纯化[J].生物工程学报,2000,16(1):74-77
[84]R R Begum,RJ Newbold,D Whitford.Purification of the membrane binding domain of cytochrome b5 by immobilised nickel chelate chromatography[J].Chromatogr.B,2000,737(122):119-130
[85]宋超,古爱国,梁洁.金属螯合亲和层析的应用研究进展[J].现代生物医学进展,2008,8(6):1178-1180
[86]韩月恒,张文红,沈岚等.Tec家族蛋白酪氨酸激酶ItkPH结构域与OS29蛋白的相互作用[J].中国生物化学与分子生物学报,2003,19(3):327-331
[87]A KMohanty,M C Wiener.Protein Exp[J].Purif.,2004,33(2):311-325
[88]UedaE.K.,GoutPW,MorgantiL.Ni(~(2+))based immobilized metalion affinity chromatography of recombinant human protachin from periplasmic Escherichia coli extracts[J].chromatogr A,2001,922(1-2):165-175
[89]Wang Xiao-jun,Fan Dai-zi.Purification of recombinant human -like collagen by using metal chelated affinity chromatography[J].Chemical Engeering of Chinese Universities,2005,3(19):410-413
[90]Greentree WK,Linder ME.Purification of recombinant G protein alpha subunits from Escherichia coli[J].Methods Mol Biol,2004,237:3-20
[91]Yang LC,Zhu Z.,Yang XG..Purification and immunity analysis of recombinant 6His-HPT protein expressed in E.coli[J].Biomed Environ,sci,2003,16(2):149-156
[92]J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南[M].北京:科学出版社,2002
[93]D.R.马歇克,J.T.门永,R.R.布格斯.蛋白质纯化与鉴定实验指南[M].北京:科学出版社,2000:78-79
[94]颜真,张英起.蛋白质研究技术[M].西安:第四军医大学出版社,2007:240
[95]郑亚军,李艳,李新菊等.不同SDS-PAGE分离胶浓度下椰子贮藏蛋白亚基的分离效果[J].热带农业科学,2008,24(9):452-456
[96]俞俊棠,唐孝宜.生物工艺学[M].上海:华东化工学院出版社,1994:90-91
[97]何成,蔡蓓蓓,楼觉人.多拷贝重组HBsAg质粒的构建及在毕赤酵母中的高效表达[J].中国生物制品学杂志,2008,21(3):207-211
[98]王瑞,杨晓野,杨莲茹等.不同碳氮源及其比例和培养基初始pH值对少孢节丛孢菌菌丝生长的影响[J].黑龙江畜牧兽医,2008,9:11-14
[99]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000
[100]范志华,张爱琳,何庆峰等.培养基影响大肠杆菌产L-天冬酰胺酶的研究[J].现代食品科技,2008,24(6):552-554
[101]朱才庆,叶勤.微量元素对大肠杆菌生长和乙酸生成的影响研究[J].微生物学报,2004,44(2):230-234
[102]张程,袁宏志.温度对发酵过程中乙醇脱氢酶的活性效应[J].山西科技,2008,4:100-104
[103]熊晓辉,于修砲,熊强等.乳酸菌发酵剂高密度培养的研究[J].中国调味品,2004(5):17-21
[104]朱凤妹,杜彬,刘长江等.β-葡萄糖苷酶产生菌摇瓶发酵条件的研究[J].食品科学,2008,29(8):422-424
[105]王海波.重组人粒细胞集落刺激因子生产工艺的研究[D].西北大学学位论文,2002:33-34
[106]申烨华.重组人γ-干扰素生产工艺优化及重组人α-糜蛋白酶复性研究[D].西北大学学位论文,2001:62-63
[107]乔世伟.微生物连续发酵过程的有关计算[J].中国调味品,2002,11(11):3-9
[108]邹运明,马武龙,高慎阳等.IPTG诱导浓度对重组李氏溶血素表达的影响[J].东北农业大学学报,2006,37(2):199-201
[109]张彩,王郡甫,刘金生等.重组人白细胞介素15工程菌高密度发酵条件探讨[J].中国生化药物杂志,2007,28(1):1-3
[110]周宇荀,曹巍,魏东芝等.抗菌肽Adenoregulin基因工程菌培养条件的优化及分批发酵研究[J].生物工程学报,2005,21(4):615-621
[111]赵春海.发酵过程的控制[J].发酵科技通讯,2007,36(4):23-25
[112]黄丽孙,玉坤黄,永红等.补料分批发酵过程控制系统研究[J].微计算机信息,2006,22(9):122-126
[113]申烨华.重组人γ-干扰素生产工艺优化及α-糜蛋白酶复性研究[D].西安:西北大学学位论文,2001
[114]崔建东,贾士儒.pH对苯丙氨酸解氨酶重组大肠杆菌生长及产酶的影响[J].食品研究与开发,2007,128(12):26-29
[115]李民,陈长庆,朴勤等.利用恒溶氧补料分批技术高密度发酵大肠杆菌生产重组人骨形成蛋白-2A[J].生物工程学报,1998,14(3):270-275
[116]颜真,张英起.蛋白质研究技术[M].西安:第四军医大学出版社,2007:95
[2]何忠平.世界制药工业发展及应对[J].医药快讯信息,2004,10(2):18-22
[3]颜真,张英起.蛋白质研究技术[M].西安:第四军医大学出版社,2007:1-2
[4]陈贯虹,迟建国,邱维忠.多肽药物的研究进展[J].山东科学,2008,21(3):42-46
[5]马楠,张英起,颜真.重组人Tum-5融合蛋白的克隆、表达、纯化及鉴定[J].第四军医大学学报,2005,26(14):1279-1281
[6]侯登勇,张英起,韩苇等.TRAIL与导向肽融合基因的克隆、表达及其表达产物的生物学活性[J].中国生物制品学杂志,2005,18(4):277-279
[7]王慧,颜真,赵宁等.RGD特异性结合多肽与重组改构人肿瘤坏死因子融合基因的克隆表达及其抑瘤活性[J].中国生物制品学杂志,2005,18(3):185-189
[8]Jieru Meng,Nan Ma,Zhen Yan,et al.NGR Enhanced the Anti-Angiogenic Activity of tum-5[J].Biochem,2006(140):1-6
[9]马楠.靶向性重组人Tum-5融合蛋白的克隆、表达、纯化及生物学活性的测定[D].西安:第四军医大学硕士学位论文,1995
[10]Folkman J..What is the evidence that tumors are angiogenesis dependent?[J].Natl Cancer Inst,1990,82(1):4-6
[11]Hanahan D.,Forlman J..Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis[J].Cell,1996,86(3):353-364
[12]Petitclerc E.,Boutaud A.,Prestayko A.,et al.New function for non-collagen type Ⅳ.Novel integrin ligands inhibting angiogenesis and tumor growth in vivo[J].Biol Chem,2000,275(11):8051-8061
[13]Maeshima Y.,Colorado PC,Torre A.,et al.Distint antitumor propertiesof a type Ⅳcollagen domain derived from basement membrane[J].Biol Chem,2000,275(28):21340-21348
[14]Maeshima Y.,Sudhakar A.,Lively JC,et al.Tumstatin,an endothelial cell-specific inhibitor of protein synthesis[J].Science,2002,295(5552):140-143
[15]Maeshima Y.,Yerramalla UL.,Dhanabal M.,et al.Extracellular matrix-derived peptide binds to alpha v beta 3 integrin and inhibits angiogenesis[J].Biol Chem,2001,276(34):31959-31968
[16]Kashtan CE.Alport syndrome and thin glomerular basement membrane disease[J].Am Soc Nephrol,1998,9(9):1736-1750
[17]Sudhakar A.,Sugimoto H.,Yang C.,et al.Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha Ⅴ beta 3 and alpha 5 beta integrins[J].Proc Natl Acad Sci USA,2003,100(8):4766-4771
[18]Maeshima Y.,Colorado PC,Kalluri R..Two RGD-independent alpha ⅴ beta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties[J].Biol Chem,2000,275(31):23745-23750
[19]顾取良,罗进贤,张添元等.人tumstatin基因的克隆、表达和抗体制备[J].中山大学学报,2005,44(2):189-192
[20]黄敏丽,罗国容,陈维平.Tumstatin肽对视网膜微血管内皮细胞增生和迁移的影响[J].眼科新进展,2008,28(9):649-652
[21]高云,俞悦,陆森.人肿瘤抑素tumstatin的基因克隆及其原核表达[J].中国生化药物杂志,2005,26(6):324-327
[22]Hamano Y.,Kalluri R..Tumstatin,the NC1 domain of a3chain of type Ⅳcollagen,is an endogenous inhibitor of pathological angiogenesis and sup resses tum or growth[J].Biochem Biophys Res Commun,2005,333(2):292-298
[23]Maeshima Y.,Manfredi M.,Reimer C.,et al.Identification of the anti-angiogenic site with in vascular basement membrane-derived tumstatin[J].Biol Chem,2001,276(18):15240-15248
[24]Netzer KO,Leinonen A.,Boutaud A..The goodpasture autoantigen.Mapping the major conformational epitope(s) of alpha3(Ⅳ) collagen to residues 17-31 and 127-141 of the NC1 domain[J].Biol Chem,1999,274(16):11267-11274
[25]Hellmark T.,Burkhardt H.,Wieslander J..Goodpasture disease.Characterization of a single conformational epitope as the target of pathogenic autoantibodies[J].Biol Chem,274(36):25862-25868
[26]任娜,王梁华,高云等.Tumstatin_(183-230)-TRAIL融合蛋白的克隆表达及其生物学双功能鉴定[J].第二军医大学学报,2008,29(5):474-478
[27]Qian Xiaohong,Xie Jianwei.The application of modern instrument analysis to the study of biomedicine[M].Beijing,Chemical Industry Press,2003:21-22
[28]Holzgrabe U.,Wawer I.,Diehl B..NMR spect roscopy in drug development and analysis [M].New York,1999:23-24
[29]Arap W.,Pasqulini R.,Ruoslahti E..Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model[J].Science,1998,279:377-380
[30]Folkman J..Angiogenesis in cancer,vascular,rheumatoid and other disease[J].Nat Med,1995,1(1):27-31
[31]Pasqualini R..Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis[J].Cancer Res,1997,15(6):722-727
[32]Curnis F.,Angelina S.,Laura B.,et al.Enhancement of tumor necrosis factor α antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N(CD13)[J].Nature Biotechnol,2000,18(2):1185-1190
[33]李民,陈常庆.重组大肠杆菌高密度发酵研究进展[J]_生物工程进展,2000,20(2):26-31
[34]孙柏欣,刘长远,陈彦.基因表达系统研究进展[J].现代农业科技,2008,2:205-208
[35]刘爽.原核系统可溶性表达策略[J].生物技术通讯,2005,16(2):172-175
[36]郭井利,彭永刚,刘鹏.重组蛋白在大肠杆菌包含体蛋白的表达及其复性[J].畜牧兽医科技信息,2005(8):65-67
[37]谢磊,孙建波,张世清.大肠杆菌表达系统及其研究进展[J].华南热带农业大学学报,2004,10(2):16-19
[38]沈毅,姚见儿,易进华等.重组大肠杆菌高密度培养的进展[J].药物生物技术,2000,7(4):243-247
[39]吴丹,仇华吉,童光志.几种表达系统的比较[J].生物技术通报,2002,2:30-34
[40]马莉.人粒细胞.巨嗜细胞集落刺激因子基因克隆,表达,摇瓶培养及分离纯化的研究[D].西安:西北大学学位论文,2004
[41]戎晶晶,刁振宇,周国华.大肠杆菌表达系统的研究进[J].药物生物技术,2005,12(6):416-420
[42]郭广君,吕素芳,王荣富.外源基因表达系统的研究进展[J].科学技术与工程,2005,6(5):582-586
[43]罗红霞,林少华,任发政.重组大肠杆菌BL21(DE3)/rbLF-N高密度培养条件优化[J].中国乳品工业,2007,35(11):10-15
[44]方方,沈佳胤,殷志敏等.乳糖诱导重组人TRA IL胞外片段在大肠杆菌中的表达[J].安徽农业大学学报,2008,35(2):266-270
[45]熊宗贵.生物技术制药[M].北京:高等教育出版社,2000,76-77
[46]DaeHyuk Kweon,Nam Soo Han,Moon Park,et al.Over 2 production of Phytolacca insularis protein in batch and fed-batch of recombinant Escherichia coli Process[J]. Biochemistry,2001,36:537-542
[47]陈航,黄晓南,陈春等.重组SOD的摇瓶发酵条件研究[J].医学理论与实践,2006,19(11):1255-1257
[48]孟文丽,许云禄.蛇毒蛋白基因的大肠杆菌表达研究进展[J].药学专论,2008,17(15):3-5
[49]吴一凡,张双全,高秀玉等.乳糖诱导pET载体表达重组蛋白的研究[J].南京师大学报,2002,25(1):89-93
[50]褚仲梅,申秀云,刘现杰等.以乳糖为诱导剂的高密度发酵[J].中国医药工业杂志,2001,32(11):491-494
[51]Betenbaugh MJ.Biotechnol[J].Bioeng,1989,33:1425-1436
[52]王瑞霞,张兆红,刘玉霞等.枯草芽孢杆菌B-903最佳发酵时间研究[J].河南农业科学,2005,3:46-48
[53]郑杭民.生物工程进展[M].北京:科学技术文献出版社,1990:135-136
[54]杨炜,王伟刚,田海英等.重组大肠杆菌高表达高密度发酵研究[J].生物技术,2006,16(3):83-86
[55]陈蔚青,陈虹,胡文浪等.重组人胰岛素样生长因子-Ⅰ工程菌高密度发酵工艺优化[J].中国生化药物杂志,2008,29(2):121-124
[56]欧俊杰.重组人干细胞因子的生产工艺研究[D].西安:西北大学学位论文,2004
[57]Riesenberg D.High-cell-density cultivation of Escherichia coli[J].Current Opinion in Biotechnology,1991,2(3):380-384
[58]Markl H.,Zenneck C.,Dubach A,et al.Cultivation or E.coli to high cell densities in a dialysis reactor[J].Microbiotechnol,1993,39(5):48-52
[59]LeeSY.High cell density culture Escherichia coli[J].TrendsBiotechnol,1996,14(3):98-105
[60]方宏清.生物工程进展[M].北京:科学出版社,1992
[61]MoriH..ChemEng[M].JPn,1979:211-214
[62]Shimizu N..FermentTeehno[M].JPn,1988:159-160
[63]倪雪朋,熊三玉,管斌.利用流加发酵进行耐氧两歧双歧杆菌高密度培养的研究[J].中国食品添加剂,2007,11(2):114-117
[64]Reisenberg D.APPI.Mierobiol[J].Bioteehol,1990,34(2):77-82
[65]张泉,于可响,郭晓宇等.重组大肠杆菌的高密度发酵放大工艺研究[J].扬州大学学报,2007,28(1):17-20
[66]乔世伟.微生物连续发酵过程的有关计算[J].中国调味品,2002,11(11):3-8
[67]王江龙,孜力汗,白凤武.絮凝酵母SPSC01连续培养最适生长条件的研究[J].微生物学报,2004,44(1):93-95
[68]刘如林.微生物工程概论[M].天津:南开大学出版社,1995:53
[69]沈萍.微生物学[M].北京:高等教育出版社,2000:135
[70]陈坚,堵国成,李寅等.发酵工程实验技术[M].北京:化学工业出版社,2003:101-102
[71]丛春水,苏志国,邓继先.恒pH葡萄糖流加方法培养重组大肠杆菌生产人肿瘤坏死因子-α及动力学研究[J].微生物学杂志,1998,18(4):44-47
[72]顾维新.重组大肠杆菌的发酵与外源基因的表达[J].海峡药学,2007,19(2):84-87
[73]艾恒.包涵体研究进展[J].中国医学文摘,2006,27(2):109-111
[74]M J Weickert,D H Doherty,E A Best,et al.Optimization of heterologous protein production in Escherichia coli[J].Currop in Biotechnol,1996,7:494-499
[75]丁兰宝,牛丹丹,石贵阳.大肠杆菌表达古菌基因的发酵条件研究[J].研究与探讨,2008,29(2):97-100
[76]霍向东,石玉瑚.大肠杆菌高密度发酵补料调控策略的研究进展[J].新疆农业科学,2004,41:16-18
[77]温进坤,韩梅.医学分子生物学[M].北京:科学出版社,1999,209-210
[78]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000,89-90
[79]D.R.马歇克,J.T.门永,R.R.布格斯.蛋白质纯化与鉴定实验指南[M].北京:科学出版社,2000:72-73
[80]骆红琴,李乃,孙文晓.一种新型金属螯合亲和色谱固定相的制备及评价[J].天津理工大学学报,2007,23(2):59-62.
[81]李蓉,陈国亮,赵文明.固定金属离子亲和色谱-蛋白质分离的方法、原理、特性和应用[J].化学通报,2005,5:352-360.
[82]Ueda E KM,Gout PW,MorgantiL[J].Chromatogr.A,2003,988:1-23.
[83]杨利,贾凌云,邹汉法.IDA型固定化镍离子金属螯合亲和膜色谱对人血清白蛋白的分离纯化[J].生物工程学报,2000,16(1):74-77
[84]R R Begum,RJ Newbold,D Whitford.Purification of the membrane binding domain of cytochrome b5 by immobilised nickel chelate chromatography[J].Chromatogr.B,2000,737(122):119-130
[85]宋超,古爱国,梁洁.金属螯合亲和层析的应用研究进展[J].现代生物医学进展,2008,8(6):1178-1180
[86]韩月恒,张文红,沈岚等.Tec家族蛋白酪氨酸激酶ItkPH结构域与OS29蛋白的相互作用[J].中国生物化学与分子生物学报,2003,19(3):327-331
[87]A KMohanty,M C Wiener.Protein Exp[J].Purif.,2004,33(2):311-325
[88]UedaE.K.,GoutPW,MorgantiL.Ni(~(2+))based immobilized metalion affinity chromatography of recombinant human protachin from periplasmic Escherichia coli extracts[J].chromatogr A,2001,922(1-2):165-175
[89]Wang Xiao-jun,Fan Dai-zi.Purification of recombinant human -like collagen by using metal chelated affinity chromatography[J].Chemical Engeering of Chinese Universities,2005,3(19):410-413
[90]Greentree WK,Linder ME.Purification of recombinant G protein alpha subunits from Escherichia coli[J].Methods Mol Biol,2004,237:3-20
[91]Yang LC,Zhu Z.,Yang XG..Purification and immunity analysis of recombinant 6His-HPT protein expressed in E.coli[J].Biomed Environ,sci,2003,16(2):149-156
[92]J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南[M].北京:科学出版社,2002
[93]D.R.马歇克,J.T.门永,R.R.布格斯.蛋白质纯化与鉴定实验指南[M].北京:科学出版社,2000:78-79
[94]颜真,张英起.蛋白质研究技术[M].西安:第四军医大学出版社,2007:240
[95]郑亚军,李艳,李新菊等.不同SDS-PAGE分离胶浓度下椰子贮藏蛋白亚基的分离效果[J].热带农业科学,2008,24(9):452-456
[96]俞俊棠,唐孝宜.生物工艺学[M].上海:华东化工学院出版社,1994:90-91
[97]何成,蔡蓓蓓,楼觉人.多拷贝重组HBsAg质粒的构建及在毕赤酵母中的高效表达[J].中国生物制品学杂志,2008,21(3):207-211
[98]王瑞,杨晓野,杨莲茹等.不同碳氮源及其比例和培养基初始pH值对少孢节丛孢菌菌丝生长的影响[J].黑龙江畜牧兽医,2008,9:11-14
[99]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000
[100]范志华,张爱琳,何庆峰等.培养基影响大肠杆菌产L-天冬酰胺酶的研究[J].现代食品科技,2008,24(6):552-554
[101]朱才庆,叶勤.微量元素对大肠杆菌生长和乙酸生成的影响研究[J].微生物学报,2004,44(2):230-234
[102]张程,袁宏志.温度对发酵过程中乙醇脱氢酶的活性效应[J].山西科技,2008,4:100-104
[103]熊晓辉,于修砲,熊强等.乳酸菌发酵剂高密度培养的研究[J].中国调味品,2004(5):17-21
[104]朱凤妹,杜彬,刘长江等.β-葡萄糖苷酶产生菌摇瓶发酵条件的研究[J].食品科学,2008,29(8):422-424
[105]王海波.重组人粒细胞集落刺激因子生产工艺的研究[D].西北大学学位论文,2002:33-34
[106]申烨华.重组人γ-干扰素生产工艺优化及重组人α-糜蛋白酶复性研究[D].西北大学学位论文,2001:62-63
[107]乔世伟.微生物连续发酵过程的有关计算[J].中国调味品,2002,11(11):3-9
[108]邹运明,马武龙,高慎阳等.IPTG诱导浓度对重组李氏溶血素表达的影响[J].东北农业大学学报,2006,37(2):199-201
[109]张彩,王郡甫,刘金生等.重组人白细胞介素15工程菌高密度发酵条件探讨[J].中国生化药物杂志,2007,28(1):1-3
[110]周宇荀,曹巍,魏东芝等.抗菌肽Adenoregulin基因工程菌培养条件的优化及分批发酵研究[J].生物工程学报,2005,21(4):615-621
[111]赵春海.发酵过程的控制[J].发酵科技通讯,2007,36(4):23-25
[112]黄丽孙,玉坤黄,永红等.补料分批发酵过程控制系统研究[J].微计算机信息,2006,22(9):122-126
[113]申烨华.重组人γ-干扰素生产工艺优化及α-糜蛋白酶复性研究[D].西安:西北大学学位论文,2001
[114]崔建东,贾士儒.pH对苯丙氨酸解氨酶重组大肠杆菌生长及产酶的影响[J].食品研究与开发,2007,128(12):26-29
[115]李民,陈长庆,朴勤等.利用恒溶氧补料分批技术高密度发酵大肠杆菌生产重组人骨形成蛋白-2A[J].生物工程学报,1998,14(3):270-275
[116]颜真,张英起.蛋白质研究技术[M].西安:第四军医大学出版社,2007:95