里氏木霉RutC-30β-甘露聚糖酶基因在大肠杆菌中的外分泌型表达
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摘要
本研究将含有β-甘露聚糖酶基因(ManI)的重组质粒pGEM-ManΙ经EcoRΙ、绿豆芽核酸酶、HindⅢ酶切处理和纯化后，获得目的基因（β-甘露聚糖酶基因）。同时，含有Ω序列和T7启动子以及信号肽OmpT序列的分泌型表达载体pT002经BamHΙ、绿豆芽核酸酶、HindⅢ酶切处理。将目的基因连接在pT002质粒信号肽OmpT之后，构建表达载体pT002-ManΙ。将表达载体pT002-ManΙ转化到大肠杆菌BL21（DE3）感受态细胞中，通过筛选获得阳性克隆子。对阳性克隆子进行PCR及酶切鉴定，并对载体中插入的目的基因进行测序。测序结果与GenBank中的里氏木霉RutC-30的β-甘露聚糖酶成熟肽cDNA序列同源性比较，结果二者序列完全一致。将表达载体pT002-ManΙ和质粒pUC18-kil共转化到大肠杆菌BL21（DE3）株，构建了外分泌型表达体系，经IPTG诱导，重组蛋白获得高效表达，SDS-聚丙烯酰胺凝胶电泳分析表明，表达的重组蛋白分子量约为51kD。将培养液浓缩纯化后，以槐豆胶为底物用3,5—二硝基水杨酸法（DNS）测定培养液中β-甘露聚糖酶的活力。纯化后的产物具有明显的β-甘露聚糖酶活性，证明重组β-甘露聚糖酶在分泌过程中得到正确加工。
In this study, the target DNA fragment of ManI gene was purified from the recombinant plasmid pGEM-ManΙ after digesting by restriction enzyme EcoRI Mung Bean Nuclease、HindⅢ, and then cloned into secretion expression vector pT002 containing Ω sequence, T7 promoter and signature sequence OmpT after digesting by restriction enzyme BamHΙ、Mung Bean Nuclease、HindⅢ. The ManI gene was fused in-frame after the signature sequence ompT in the resulting plasmid pT002-ManΙ. The positive clones were screened in E.coli BL21 transformed by the recombinant plasmid and then subjected to sequencing after identification by PCR. The sequence of cloned ManI gene are completely identical to corresponding cDNA sequences of Trichoderma Reesei Rut- C30 of β-Mannanase mature peptides. The secretion expression system was established with E.coli BL21 co-transformed by pT002-ManΙ and pUC18-kil according to the principle kil can enhance the permeabilization of cell membrane and promote the secretion of periplasmic protein. The fusion β-Mannanase with molecular weights of about 51 kD as indicated by SDS-PAGE analysis was expressed with high-efficency after inducing by IPTG. The activity of β-Mannanasein in the concentrated medium was determined with locost bean gum as substract by 3,5-dinitosalicylic acid method, and the result indicated that the purified product showed apparentβ-Mannanase activity and the recombinant β-Mannanase was correctly processed during the secretion.
In this study, the target DNA fragment of ManI gene was purified from the recombinant plasmid pGEM-ManΙ after digesting by restriction enzyme EcoRI Mung Bean Nuclease、HindⅢ, and then cloned into secretion expression vector pT002 containing Ω sequence, T7 promoter and signature sequence OmpT after digesting by restriction enzyme BamHΙ、Mung Bean Nuclease、HindⅢ. The ManI gene was fused in-frame after the signature sequence ompT in the resulting plasmid pT002-ManΙ. The positive clones were screened in E.coli BL21 transformed by the recombinant plasmid and then subjected to sequencing after identification by PCR. The sequence of cloned ManI gene are completely identical to corresponding cDNA sequences of Trichoderma Reesei Rut- C30 of β-Mannanase mature peptides. The secretion expression system was established with E.coli BL21 co-transformed by pT002-ManΙ and pUC18-kil according to the principle kil can enhance the permeabilization of cell membrane and promote the secretion of periplasmic protein. The fusion β-Mannanase with molecular weights of about 51 kD as indicated by SDS-PAGE analysis was expressed with high-efficency after inducing by IPTG. The activity of β-Mannanasein in the concentrated medium was determined with locost bean gum as substract by 3,5-dinitosalicylic acid method, and the result indicated that the purified product showed apparentβ-Mannanase activity and the recombinant β-Mannanase was correctly processed during the secretion.
引文
Bouh KC, Shareck F, Dea S .Monoclonal Antibodies to Escherichia coli-Expressed P46 and P65 Membranous Proteins for Specific Immunodetection of Mycoplasma hyopneumoniae in Lungs of Infected Pigs. Clin Diagn Lab Immunol. 2003 May;10(3):459-68.
Yin Y, Lu H, Khosla C, Cane DE. Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase. J Am Chem Soc. 2003 May 14;125(19):5671-6.
Mori S, Akao S, Nankai H, Hashimoto W, Mikami B, Murata K. A novel member of glycoside hydrolase family 88: overexpression, purification, and characterization of unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1. Protein Expresstion Purification. 2003 May;29(1):77-84.
Fu Z, Hamid SB, Razak CN, Basri M, Salleh AB, Rahman RN. Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease. Protein Expr Purif. 2003 Mar;28(1):63-8.
Kirkpatrick RB, McDevitt PJ, Matico RE, Nwagwu S, Trulli SH, Mao J, Moore DD, Yorke AF, McLaughlin MM, Knecht KA, Elefante LC, Calamari AS, Fornwald JA, Trill JJ, Jonak ZL, Kane J, Patel PS, Sathe GM, Shatzman AR, Tapley PM, Johanson KO. A bicistronic expression system for bacterial production of authentic human interleukin-18. Protein Expr Purif. 2003 Feb;27(2):279-92.
T. R. Tuler, M. J. Callanan and T. R. Klaenhammer .Overexpression of Peptidases in Lactococcus and Evaluation of Their Release from Leaky Cells J. Dairy Sci. 85:2438-2450
Humphries HE, Christodoulides M, Heckels JE. Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag. Protein Expr Purif. 2002 Nov;26(2):243-8.
Zhang F, Zhou D, Kao YC, Ye J, Chen S. Expression and purification of a recombinant form of human aromatase from Escherichia coli. Biochem Pharmacol. 2002 Nov 1;64(9):1317-24.
Tong Q, Zhang HT, Yang YG, Yang SL, Gong Y. An Expression Release System for Heterogeneous Proteins in E.coli Mediated by the kil Gene. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2001;33(5):537-541.
Park SJ, Lee SK, Lee BJ. Effect of tandem rare codon substitution and vector-host combinations on the expression of the EBV gp110 C-terminal domain in Escherichia coli. Protein Expr Purif. 2002 Apr;24(3):470-80.
Ma C, Marassi FM, Jones DH, Straus SK, Bour S, Strebel K, Schubert U, Oblatt-Montal M, Montal M, Opella SJ. Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1. Protein Sci. 2002 Mar;11(3):546-57.
Qiao H, Zhao M, Yuan X. Expression of heat shock protein 90 alpha and its effect on the growth of tumor in mice Zhonghua Zhong Liu Za Zhi. 1999 Jan;21(1):19-21.
Montell E, Lerin C, Newgard CB, Gomez-Foix AM. Effects of modulation of glycerol kinase expression on lipid and carbohydrate metabolism in human muscle cells. J Biol Chem. 2002 Jan 25;277(4):2682-6. Epub 2001 Nov 19.
Cartee RT, Forsee WT, Jensen JW, Yother J. Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer. J Biol Chem. 2001 Dec 28;276(52):48831-9. Epub 2001 Oct 29.
Chisholm J, Wieczorek A, Sanfacon H. Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor. Virus Res. 2001 Nov 5;79(1-2):153-64.
Szweda P, Pladzyk R, Kotlowski R, Kur J. Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system. Protein Expr Purif. 2001 Aug;22(3):467-71.
Lee J, Filosa S, Bonvin J, Guyon S, Aponte RA, Turnbull JL. Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli. Protein Expr Purif. 2001 Jul;22(2):180-8.
Morita M, Asami K, Tanji Y, Unno H. Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes. Biotechnol Prog. 2001 May-Jun;17(3):573-6
Kosovsky J, Durmanova V, Kudelova M, Rezuchova I, Tkacikova L, Rajcani J. A simple procedure for expression and purification of selected non-structural (alpha and beta) herpes simplex virus 1 (HSV-1) proteins. J Virol Methods. 2001 Apr;92(2):121-9.
Trepod CM, Mott JE. Modification of the carboxy-terminal amino acid sequence alters the Escherichia coli expression of a gene encoding multiple repeats of a bovine growth hormone releasing factor analog. J Biotechnol. 2000 Dec 28;84(3):273-84.
Valentin E, Singer AG, Ghomashchi F, Lazdunski M, Gelb MH, Lambeau G. Cloning and recombinant expression of human group IIF-secreted phospholipase A(2). Biochem Biophys Res Commun. 2000 Dec 9;279(1):223-8.
Roldan-Arjona T, Garcia-Ortiz MV, Ruiz-Rubio M, Ariza RR. Plant Mol Biol.cDNA cloning, expression and functional characterization of an Arabidopsis thaliana homologue of the Escherichia coli DNA repair enzyme endonuclease III. 2000 Sep;44(1):43-52.
Halstead JR, Fransen MP, Eberhart RY, Park AJ, Gilbert HJ, Hazlewood GP. alpha-Galactosidase A from Pseudomonas fluorescens subsp. cellulosa: cloning, high level expression and its role in galactomannan hydrolysis. FEMS Microbiol Lett. 2000 Nov 15;192(2):197-203.
Gavit P, Better M. Production of antifungal recombinant peptides in Escherichia coli. J Biotechnol. 2000 Apr 28;79(2):127-36.
Li M, Li LY, Wu X, Liang SP. Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider (Selenocosmia huwena). Toxicon. 2000 Feb;38(2):153-62.
Karamyshev AL, Karamysheva ZN, Ito K, Matsufuji S, Nakamura Y. Overexpression and purification of recombinant eRF1 proteins of rabbit and Tetrahymena thermophila. Biochemistry (Mosc). 1999 Dec;64(12):1391-400.
Oh GH, Hahm MS, Chung BH. Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli. Protein Expr Purif. 1999 Dec;17(3):428-34.
Dharmawardhana DP, Ellis BE, Carlson JE. cDNA cloning and heterologous expression of coniferin beta-glucosidase.Plant Mol Biol. 1999 May;40(2):365-72.
Kawamoto S, Mizuguchi Y, Morimoto K, Aki T, Shigeta S, Yasueda H, Wada T, Suzuki O, Jyo T, Ono K. Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae. Biochim Biophys Acta. 1999 Jul 7;1454(2):201-7.
Eibensteiner P, Denepoux S, Steinberger P, Kraft D, Visco V, Banchereau J, Lebecque S, Valenta R. Expression of a human IgG4 antibody, BAB2, with specificity for the major Birch pollen allergen, Bet v 1 in Escherichia coli: recombinant BAB2 Fabs enhance the allergic reaction. Int Arch Allergy Immunol. 1999 Feb-Apr;118(2-4):190-2.
Yoshikawa F, Uchiyama T, Iwasaki H, Tomomori-Satoh C, Tanaka T, Furuichi T, Mikoshiba K. High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli. Biochem Biophys Res Commun. 1999 Apr 21;257(3):792-7.
Kutyrev V, Mehigh RJ, Motin VL, Pokrovskaya MS, Smirnov GB, Brubaker RR. Expression of the plague plasminogen activator in Yersinia pseudotuberculosis and Escherichia coli. Infect Immun. 1999 Mar;67(3):1359-67.
Rippmann JF, Klein M, Hoischen C, Brocks B, Rettig WJ, Gumpert J, Pfizenmaier K, Mattes R, Moosmayer D. Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli. Appl Environ Microbiol. 1998 Dec;64(12):4862-9.
Pathak N, Hu SI, Koehn JA. The expression, refolding, and purification of the catalytic domain of human collagenase-3 (MMP-13). Protein Expr Purif. 1998 Nov;14(2):283-8.
Kim YO, Lee JK, Kim HK, Yu JH, Oh TK. Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbiol Lett. 1998 May 1;162(1):185-91.
Scheurer S, Metzner K, Haustein D, Vieths S. Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen. Mol Immunol. 1997 Jun;34(8-9):619-29.
Chen L, Thompson NL, Pielak GJ. Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions. Protein Sci. 1997 May;6(5):1038-46.
Miksch G, Fiedler E, Dobrowolski P, Friehs K. The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phaseArch Microbiol. 1997 Mar 7;167(2/3):143-50.
Miksch G, Kleist S, Friehs K, Flaschel E. Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors. Appl Microbiol Biotechnol. 2002 Sep;59(6):685-94. Epub 2002 Jul 17.
Tanaka T, Horio T, Matuo Y. Secretory production of recombinant human C-reactive protein in Escherichia coli, capable of binding with phosphorylcholine, and its characterization. Biochem Biophys Res Commun. 2002 Jul 5;295(1):163-6.
Ptitsyn LR, Altman IB. [Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells] Bioorg Khim. 1999 Dec;25(12):923-9.
Miksch G, Neitzel R, Friehs K, Flaschel E. High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium. Appl Microbiol Biotechnol. 1999 May;51(5):627-32.
Miksch G, Fiedler E, Dobrowolski P, Flaschel E. Controlled secretion into the culture medium of a hybrid beta-glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5-derived transposon. Appl Microbiol Biotechnol. 1997 May;47(5):530-6.
Robbens J, Raeymaekers A, Steidler L, Fiers W, Remaut E. Production of soluble and active recombinant murine interleukin-2 in Escherichia coli: high level expression, Kil-induced release, and purification. Protein Expr Purif. 1995 Aug;6(4):481-6.
Aono R. Release of penicillinase by Escherichia coli HB101 (pEAP31) accompanying the simultaneous release of outer-membrane components by Kil peptide. Biochem J. 1989 Oct 1;263(1):65-71.
Blanchin-Roland S, Masson JM. Protein secretion controlled by a synthetic gene in Escherichia coli. Protein Eng. 1989 Mar;2(6):473-80.
Kato H, Kato C, Yanagida T, Tanaka Y, Moriyama S, Sasaki K, Kudo T, Horikoshi K. Construction of a secretion vector production of peptide hormones in Escherichia coli (extracellular production of calcitonin as fused protein). FEMS Microbiol Lett. 1989 Feb;48(3):339-44.
Suit JL, Luria SE. Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death. J Bacteriol. 1988 Oct;170(10):4963-6.
Kobayashi T, Kato C, Kudo T, Horikoshi K. Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9. J Bacteriol. 1986 Jun;166(3):728-32.
Ignatova Z, Mahsunah A, Georgieva M, Kasche V. Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains. Appl Environ Microbiol. 2003 Feb;69(2):1237-45.
Matuo Y, Tanaka T, Kagei M, Sugimoto M. [Production of recombinant human CRP and its application on clinical testing] Rinsho Byori. 2002 Jan;50(1):13-9.
Sergueev K, Yu D, Austin S, Court D. Cell toxicity caused by products of the p(L) operon of bacteriophage lambda. Gene. 2001 Jul 11;272(1-2):227-35.
Hagglund P, Eriksson T, Collen A, Nerinckx W, Claeyssens M, Stalbrand H. A cellulose-binding module of the Trichoderma reesei beta-mannanase Man5A increases the mannan-hydrolysis of complex substrates. J Biotechnol. 2003 Feb 27;101(1):37-48.
Xu B, Sellos D, Janson JC. Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) beta-mannanase gene. Eur J Biochem. 2002 Mar;269(6):1753-60.
Xu B, Munoz I IG, Janson JC, Stahlberg J. Crystallization and X-ray analysis of native and selenomethionyl beta-mannanase Man5A from blue mussel, Mytilus edulis, expressed in Pichia pastoris. Acta Crystallogr D Biol Crystallogr. 2002 Mar;58(Pt 3):542-5.
Setati ME, Ademark P, van Zyl WH, Hahn-Hagerdal B, Stalbrand H. Expression of the Aspergillus aculeatus endo-beta-1,4-mannanase encoding gene (man1) in Saccharomyces cerevisiae and characterization of the recombinant enzyme.Protein Expr Purif. 2001 Feb;21(1):105-14.
Gibbs MD, Reeves RA, Sunna A, Bergquist PL. Sequencing and expression of a beta-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme. Curr Microbiol. 1999 Dec;39(6):351-0357.
Cann IK, Kocherginskaya S, King MR, White BA, Mackie RI. Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene from Thermoanaerobacterium polysaccharolyticum. J Bacteriol. 1999 Mar;181(5):1643-51.
Ethier N, Talbot G, Sygusch J. Gene cloning, DNA sequencing, and expression of thermostable beta-mannanase from Bacillus stearothermophilus. Appl Environ Microbiol. 1998 Nov;64(11):4428-32.
Bewley JD, Burton RA, Morohashi Y, Fincher GB. Molecular cloning of a cDNA encoding a (1-->4)-beta-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum). Planta. 1997 Dec;203(4):454-9.
Gibbs MD, Elinder AU, Reeves RA, Bergquist PL. Sequencing, cloning and expression of a beta-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4. FEMS Microbiol Lett. 1996 Jul 15;141(1):37-43.
Stalbrand H, Saloheimo A, Vehmaanpera J, Henrissat B, Penttila M. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain. Appl Environ Microbiol. 1995 Mar;61(3):1090-7.
Christgau S, Kauppinen S, Vind J, Kofod LV, Dalboge H. Expression cloning, purification and characterization of a beta-1,4-mannanase from Aspergillus aculeatus. Biochem Mol Biol Int. 1994 Aug;33(5):917-25.
Luthi E, Jasmat NB, Grayling RA, Love DR, Bergquist PL. Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum".Appl Environ Microbiol. 1991 Mar;57(3):694-700.
Talbot G, Sygusch J. Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus. Appl Environ Microbiol. 1990 Nov;56(11):3505-10.
Mandels,M et al. Problems and challenges in the cellulose to cellulase fermenation. Proc Biochem . 1978， 13;6-13
罗文新 夏宁邵 一种带增强子的原核高效表达载体的构建及初步应用 生物工程学报 ，2000，16卷5期，578-581
毕群 黄仪秀 朱圣庚 水蛭素基因的递归PCR合成和在大肠杆菌中的表达与分泌 北京大学学报（自然科学版），1997，第33卷，第6期
龙建银 王会信 刘莉 王芳 周廷冲 人转化生长因子β1在大肠杆菌中的分泌表达 中国生物化学与分子生物学学报，第15卷，第2期
袁涛 张雪梅 黄林 大肠杆菌分泌型表达重组人碱性成纤维细胞生长因子 华西药学杂志，1999，14（5-6），319-322
张同海 赵为诚 陈昆明 鲑鱼生长激素基因分泌型表达质粒的构建 生物化学与生物物理学进展，1994，21（4）
张宏权 王允玲 周廷冲 大肠杆菌分泌表达载体的构建和人表皮生长因子在大肠杆菌中的分泌性表达 生物化学杂志， 1995，第11卷第４期
黄仪秀 陈杰鹏 毕群 朱圣庚 应用PCR重组技术构建大肠杆菌分泌型表达载体 生物化学杂志，1995，第11卷，第6期
李军 李伯良 蛋白A信号肽引导的E.coli外泌高表达异源蛋白 生物化学与生物物理学报，1995，第27卷，第6期
宋礼华 桂向东 范清林 分泌型重组人生长激素的研究 生物学杂志，1999，Vol.16 No.1
邓建蓓 陈萍 王雨岭 带有蛋白标签的原核表达载体构建第四军医大学学报，1998，19（3）
刘永亮 童克忠 利用枯草杆菌碱性蛋白酶E基因的信号顺序构建分泌型表达载体 遗传学报 1994，21（3） 235-246
陈燕 李金照 夏令朝 人胰岛素原基因在大肠杆菌中高效外分泌表达 生物化学杂志 1995，Vol.11 No.6
蒙健宗 李晓明 王青艳 高密度培养基因工程大肠杆菌生产 α-乙酰乳酸脱羧酶 广西科学 2001，8（4）284-286，290
牛利国 陈志南 苏成芝 药立波抗 人肝癌单链抗体的基因克隆和分泌型表达细胞与分子免疫学杂志 1998，14（2） 102-109
刘纯杰 张兆山 李淑琴 黄翠芹幽门螺杆菌热休克蛋白A基因的克隆及高效分泌 表达军事医学科学院院刊 2000，9 Vol24 No.3 161-165
贺华君 杨卫东 施惠娟 吴祥甫 分泌癌胚抗原肿瘤靶向性Cu,Zn-SOD的基因克隆基因表达 华东理工大学学报 2001，4，Vol27 No.2 124-128
叶传忠 关明 张芳林 苏兵 分泌型肿瘤坏死因子相关激活诱导因子融合蛋白在大肠杆菌中的表达和纯化 复旦学报（医学科学版） 2001，7 28（4）
郭丽英 钟晓燕 吴祥甫 江苏蝮蛇神经生长因子在大肠杆菌中的表达及纯化生物工程学报 1999，7 Vol 15 No.3
梁爱华 苏智广 李晓玲 东亚钳蝎神经毒素在大肠杆菌中的表达 中国生物化学与分子生物学报 1999，4 Vo l5 No.2 205-210
赵宝华 许崇波 抗ａ型产气荚膜α菌毒素单链抗体基因的克隆和表达 生物工程学报 2001，9 Vo 17 No.5
江培诩 钱志康 黄伟达 大肠杆菌aroG基因在枯草杆菌中的分泌表达复旦学报(自然科学版) 2000,6 Vol 39 No.3
杨运桂 童芹 郑卫东 分子伴侣过量表达对蛋白质分泌及可溶性的影响 中国生物化学与分子生物学报 2000，6， Vol 16 No.3 382-387
盛涛 张建中 刘延清 利用自身ctxb启动子表达CTB 微生物免疫学进展 2001，Vol 29 No.4
韩照中 金明 冯尔玲 L-天门冬酰胺酶的可溶性表达研究 微生物学免疫学进展 2000，28（3）
王戈林 沈萍 杨澜 嗜麦芽假单胞菌酪氨酸酶基因在大肠杆菌中的克隆与表达 遗传学报 1999，26（3）274-279
陈建军 曹军卫 苏莉嗜酸嗜碱芽孢杆菌NTT76启动子和信号肽序列的克隆及表达 武汉大学学报理学版 2001，8 Vol 47 No.4 471-476
李武平 王志武 朱丽娜 张雪梅 刘丽军 分泌型重组人生长激素工程菌的高密度发酵 中国生物制品学杂志 2000，Vol 13 No.3、
唐雪明 邵蔚蓝 沈微 王正祥 枯草杆菌6816碱性蛋白酶基因在大肠杆菌中的克隆和表达 生物技术 2001，8 Vol 11 No.4
席全胜 钱欣国 周庆玮 潘巍人 epiregulin 在大肠杆菌中的表达 生物化学与生物物理学报 2000，32（3）295-298
张菊 颜真 韩苇 王俊楼 Angiostatin 基因的克隆及在原核细胞中的表达第四军医大学学报 1999，20（7） 607-612
安云庆 柯岩 杨贵贞 BPI23-Fcγ1重组蛋白原核表达的载体的构建、表达和生物学活性的测定 中国医学科学院学报 2000，Vol 22 No.6 511-516
林琳 任凤 赵宝昌 金晓艳 Balb/C小鼠金属硫蛋白基因-1在大肠杆菌中的高效表达 药物生物技术 2001，8（3）：139-142
王朝健 李元 CTLA-4在大肠杆菌中的克隆和表达 中国医学科学院学报 2001，4 Vol 23 No.2
李节 邱平铺，葡萄球菌蛋白A分泌型融合表达系统 国外医学、预防、诊断、治疗生物制品分册 1994，Vol 17 No.5 193-196
韩苇 颜真 王俊楼 赵永同 EPO模拟肽基因4串联体的构建和表达 第四军医大学学报 2001，22（4） 319-321
吴乃虎 基因工程原理 科学出版社 2001
卢圣栋 现代分子生物学实验技术（第二版） 中国协和医科大学出版社 1999
北京化学试剂公司 化学试剂手册 北京工业大学出版社 1993
方德福 周吕 丁濂 张德昌 现代医学实验技巧丛书 北京医科大学中国协和医科大学联合出版社 1996
彭秀玲 袁汉英 谢毅 王洪海基因工程实验技术 湖南科学技术出版社
朱玉贤 李毅 现代分子生物学 高等教育出版社 1998
周德庆 微生物学教程及实验方法 高等教育出版社
张龙翔 生物化学实验技术与方法 科学出版社
林万明 PCR实验技术指南 科学出版社
C.W.迪芬巴赫 G.S.德维克斯勒 著 黄培党 俞炜源 陈添弥 译 PCR技术实验指南 科学出版社 1998
邬敏辰 β-甘露聚糖酶及其水解产物的应用研究 江苏调味副食品 2001年(总第69期) 5-8
杨巍 张岚 卢智刚 翟中和 利用两种不相容质粒在大肠杆菌中共表达DEF45和DEF40 生物化学与生物物理学报 2001,33(2)：238-242
高宏斌 里氏木霉RUT—C30 β—甘露聚糖酶的诱导表达及其cDNA的克隆 内蒙古农业大学硕士学位论文 2003
范文斌 王龙 王和平 抗甘露聚糖酶多克隆抗体的制备及其ELISA双抗夹心法的建立 内蒙古农业大学学报 2004，24（4）89-93
Yin Y, Lu H, Khosla C, Cane DE. Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase. J Am Chem Soc. 2003 May 14;125(19):5671-6.
Mori S, Akao S, Nankai H, Hashimoto W, Mikami B, Murata K. A novel member of glycoside hydrolase family 88: overexpression, purification, and characterization of unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1. Protein Expresstion Purification. 2003 May;29(1):77-84.
Fu Z, Hamid SB, Razak CN, Basri M, Salleh AB, Rahman RN. Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease. Protein Expr Purif. 2003 Mar;28(1):63-8.
Kirkpatrick RB, McDevitt PJ, Matico RE, Nwagwu S, Trulli SH, Mao J, Moore DD, Yorke AF, McLaughlin MM, Knecht KA, Elefante LC, Calamari AS, Fornwald JA, Trill JJ, Jonak ZL, Kane J, Patel PS, Sathe GM, Shatzman AR, Tapley PM, Johanson KO. A bicistronic expression system for bacterial production of authentic human interleukin-18. Protein Expr Purif. 2003 Feb;27(2):279-92.
T. R. Tuler, M. J. Callanan and T. R. Klaenhammer .Overexpression of Peptidases in Lactococcus and Evaluation of Their Release from Leaky Cells J. Dairy Sci. 85:2438-2450
Humphries HE, Christodoulides M, Heckels JE. Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag. Protein Expr Purif. 2002 Nov;26(2):243-8.
Zhang F, Zhou D, Kao YC, Ye J, Chen S. Expression and purification of a recombinant form of human aromatase from Escherichia coli. Biochem Pharmacol. 2002 Nov 1;64(9):1317-24.
Tong Q, Zhang HT, Yang YG, Yang SL, Gong Y. An Expression Release System for Heterogeneous Proteins in E.coli Mediated by the kil Gene. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2001;33(5):537-541.
Park SJ, Lee SK, Lee BJ. Effect of tandem rare codon substitution and vector-host combinations on the expression of the EBV gp110 C-terminal domain in Escherichia coli. Protein Expr Purif. 2002 Apr;24(3):470-80.
Ma C, Marassi FM, Jones DH, Straus SK, Bour S, Strebel K, Schubert U, Oblatt-Montal M, Montal M, Opella SJ. Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1. Protein Sci. 2002 Mar;11(3):546-57.
Qiao H, Zhao M, Yuan X. Expression of heat shock protein 90 alpha and its effect on the growth of tumor in mice Zhonghua Zhong Liu Za Zhi. 1999 Jan;21(1):19-21.
Montell E, Lerin C, Newgard CB, Gomez-Foix AM. Effects of modulation of glycerol kinase expression on lipid and carbohydrate metabolism in human muscle cells. J Biol Chem. 2002 Jan 25;277(4):2682-6. Epub 2001 Nov 19.
Cartee RT, Forsee WT, Jensen JW, Yother J. Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer. J Biol Chem. 2001 Dec 28;276(52):48831-9. Epub 2001 Oct 29.
Chisholm J, Wieczorek A, Sanfacon H. Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor. Virus Res. 2001 Nov 5;79(1-2):153-64.
Szweda P, Pladzyk R, Kotlowski R, Kur J. Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system. Protein Expr Purif. 2001 Aug;22(3):467-71.
Lee J, Filosa S, Bonvin J, Guyon S, Aponte RA, Turnbull JL. Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli. Protein Expr Purif. 2001 Jul;22(2):180-8.
Morita M, Asami K, Tanji Y, Unno H. Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes. Biotechnol Prog. 2001 May-Jun;17(3):573-6
Kosovsky J, Durmanova V, Kudelova M, Rezuchova I, Tkacikova L, Rajcani J. A simple procedure for expression and purification of selected non-structural (alpha and beta) herpes simplex virus 1 (HSV-1) proteins. J Virol Methods. 2001 Apr;92(2):121-9.
Trepod CM, Mott JE. Modification of the carboxy-terminal amino acid sequence alters the Escherichia coli expression of a gene encoding multiple repeats of a bovine growth hormone releasing factor analog. J Biotechnol. 2000 Dec 28;84(3):273-84.
Valentin E, Singer AG, Ghomashchi F, Lazdunski M, Gelb MH, Lambeau G. Cloning and recombinant expression of human group IIF-secreted phospholipase A(2). Biochem Biophys Res Commun. 2000 Dec 9;279(1):223-8.
Roldan-Arjona T, Garcia-Ortiz MV, Ruiz-Rubio M, Ariza RR. Plant Mol Biol.cDNA cloning, expression and functional characterization of an Arabidopsis thaliana homologue of the Escherichia coli DNA repair enzyme endonuclease III. 2000 Sep;44(1):43-52.
Halstead JR, Fransen MP, Eberhart RY, Park AJ, Gilbert HJ, Hazlewood GP. alpha-Galactosidase A from Pseudomonas fluorescens subsp. cellulosa: cloning, high level expression and its role in galactomannan hydrolysis. FEMS Microbiol Lett. 2000 Nov 15;192(2):197-203.
Gavit P, Better M. Production of antifungal recombinant peptides in Escherichia coli. J Biotechnol. 2000 Apr 28;79(2):127-36.
Li M, Li LY, Wu X, Liang SP. Cloning and functional expression of a synthetic gene encoding huwentoxin-I, a neurotoxin from the Chinese bird spider (Selenocosmia huwena). Toxicon. 2000 Feb;38(2):153-62.
Karamyshev AL, Karamysheva ZN, Ito K, Matsufuji S, Nakamura Y. Overexpression and purification of recombinant eRF1 proteins of rabbit and Tetrahymena thermophila. Biochemistry (Mosc). 1999 Dec;64(12):1391-400.
Oh GH, Hahm MS, Chung BH. Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli. Protein Expr Purif. 1999 Dec;17(3):428-34.
Dharmawardhana DP, Ellis BE, Carlson JE. cDNA cloning and heterologous expression of coniferin beta-glucosidase.Plant Mol Biol. 1999 May;40(2):365-72.
Kawamoto S, Mizuguchi Y, Morimoto K, Aki T, Shigeta S, Yasueda H, Wada T, Suzuki O, Jyo T, Ono K. Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae. Biochim Biophys Acta. 1999 Jul 7;1454(2):201-7.
Eibensteiner P, Denepoux S, Steinberger P, Kraft D, Visco V, Banchereau J, Lebecque S, Valenta R. Expression of a human IgG4 antibody, BAB2, with specificity for the major Birch pollen allergen, Bet v 1 in Escherichia coli: recombinant BAB2 Fabs enhance the allergic reaction. Int Arch Allergy Immunol. 1999 Feb-Apr;118(2-4):190-2.
Yoshikawa F, Uchiyama T, Iwasaki H, Tomomori-Satoh C, Tanaka T, Furuichi T, Mikoshiba K. High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli. Biochem Biophys Res Commun. 1999 Apr 21;257(3):792-7.
Kutyrev V, Mehigh RJ, Motin VL, Pokrovskaya MS, Smirnov GB, Brubaker RR. Expression of the plague plasminogen activator in Yersinia pseudotuberculosis and Escherichia coli. Infect Immun. 1999 Mar;67(3):1359-67.
Rippmann JF, Klein M, Hoischen C, Brocks B, Rettig WJ, Gumpert J, Pfizenmaier K, Mattes R, Moosmayer D. Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli. Appl Environ Microbiol. 1998 Dec;64(12):4862-9.
Pathak N, Hu SI, Koehn JA. The expression, refolding, and purification of the catalytic domain of human collagenase-3 (MMP-13). Protein Expr Purif. 1998 Nov;14(2):283-8.
Kim YO, Lee JK, Kim HK, Yu JH, Oh TK. Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbiol Lett. 1998 May 1;162(1):185-91.
Scheurer S, Metzner K, Haustein D, Vieths S. Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen. Mol Immunol. 1997 Jun;34(8-9):619-29.
Chen L, Thompson NL, Pielak GJ. Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions. Protein Sci. 1997 May;6(5):1038-46.
Miksch G, Fiedler E, Dobrowolski P, Friehs K. The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phaseArch Microbiol. 1997 Mar 7;167(2/3):143-50.
Miksch G, Kleist S, Friehs K, Flaschel E. Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors. Appl Microbiol Biotechnol. 2002 Sep;59(6):685-94. Epub 2002 Jul 17.
Tanaka T, Horio T, Matuo Y. Secretory production of recombinant human C-reactive protein in Escherichia coli, capable of binding with phosphorylcholine, and its characterization. Biochem Biophys Res Commun. 2002 Jul 5;295(1):163-6.
Ptitsyn LR, Altman IB. [Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells] Bioorg Khim. 1999 Dec;25(12):923-9.
Miksch G, Neitzel R, Friehs K, Flaschel E. High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium. Appl Microbiol Biotechnol. 1999 May;51(5):627-32.
Miksch G, Fiedler E, Dobrowolski P, Flaschel E. Controlled secretion into the culture medium of a hybrid beta-glucanase by Acetobacter methanolicus mediated by the kil gene of Escherichia coli located on a Tn5-derived transposon. Appl Microbiol Biotechnol. 1997 May;47(5):530-6.
Robbens J, Raeymaekers A, Steidler L, Fiers W, Remaut E. Production of soluble and active recombinant murine interleukin-2 in Escherichia coli: high level expression, Kil-induced release, and purification. Protein Expr Purif. 1995 Aug;6(4):481-6.
Aono R. Release of penicillinase by Escherichia coli HB101 (pEAP31) accompanying the simultaneous release of outer-membrane components by Kil peptide. Biochem J. 1989 Oct 1;263(1):65-71.
Blanchin-Roland S, Masson JM. Protein secretion controlled by a synthetic gene in Escherichia coli. Protein Eng. 1989 Mar;2(6):473-80.
Kato H, Kato C, Yanagida T, Tanaka Y, Moriyama S, Sasaki K, Kudo T, Horikoshi K. Construction of a secretion vector production of peptide hormones in Escherichia coli (extracellular production of calcitonin as fused protein). FEMS Microbiol Lett. 1989 Feb;48(3):339-44.
Suit JL, Luria SE. Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death. J Bacteriol. 1988 Oct;170(10):4963-6.
Kobayashi T, Kato C, Kudo T, Horikoshi K. Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9. J Bacteriol. 1986 Jun;166(3):728-32.
Ignatova Z, Mahsunah A, Georgieva M, Kasche V. Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains. Appl Environ Microbiol. 2003 Feb;69(2):1237-45.
Matuo Y, Tanaka T, Kagei M, Sugimoto M. [Production of recombinant human CRP and its application on clinical testing] Rinsho Byori. 2002 Jan;50(1):13-9.
Sergueev K, Yu D, Austin S, Court D. Cell toxicity caused by products of the p(L) operon of bacteriophage lambda. Gene. 2001 Jul 11;272(1-2):227-35.
Hagglund P, Eriksson T, Collen A, Nerinckx W, Claeyssens M, Stalbrand H. A cellulose-binding module of the Trichoderma reesei beta-mannanase Man5A increases the mannan-hydrolysis of complex substrates. J Biotechnol. 2003 Feb 27;101(1):37-48.
Xu B, Sellos D, Janson JC. Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) beta-mannanase gene. Eur J Biochem. 2002 Mar;269(6):1753-60.
Xu B, Munoz I IG, Janson JC, Stahlberg J. Crystallization and X-ray analysis of native and selenomethionyl beta-mannanase Man5A from blue mussel, Mytilus edulis, expressed in Pichia pastoris. Acta Crystallogr D Biol Crystallogr. 2002 Mar;58(Pt 3):542-5.
Setati ME, Ademark P, van Zyl WH, Hahn-Hagerdal B, Stalbrand H. Expression of the Aspergillus aculeatus endo-beta-1,4-mannanase encoding gene (man1) in Saccharomyces cerevisiae and characterization of the recombinant enzyme.Protein Expr Purif. 2001 Feb;21(1):105-14.
Gibbs MD, Reeves RA, Sunna A, Bergquist PL. Sequencing and expression of a beta-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme. Curr Microbiol. 1999 Dec;39(6):351-0357.
Cann IK, Kocherginskaya S, King MR, White BA, Mackie RI. Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene from Thermoanaerobacterium polysaccharolyticum. J Bacteriol. 1999 Mar;181(5):1643-51.
Ethier N, Talbot G, Sygusch J. Gene cloning, DNA sequencing, and expression of thermostable beta-mannanase from Bacillus stearothermophilus. Appl Environ Microbiol. 1998 Nov;64(11):4428-32.
Bewley JD, Burton RA, Morohashi Y, Fincher GB. Molecular cloning of a cDNA encoding a (1-->4)-beta-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum). Planta. 1997 Dec;203(4):454-9.
Gibbs MD, Elinder AU, Reeves RA, Bergquist PL. Sequencing, cloning and expression of a beta-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4. FEMS Microbiol Lett. 1996 Jul 15;141(1):37-43.
Stalbrand H, Saloheimo A, Vehmaanpera J, Henrissat B, Penttila M. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain. Appl Environ Microbiol. 1995 Mar;61(3):1090-7.
Christgau S, Kauppinen S, Vind J, Kofod LV, Dalboge H. Expression cloning, purification and characterization of a beta-1,4-mannanase from Aspergillus aculeatus. Biochem Mol Biol Int. 1994 Aug;33(5):917-25.
Luthi E, Jasmat NB, Grayling RA, Love DR, Bergquist PL. Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum".Appl Environ Microbiol. 1991 Mar;57(3):694-700.
Talbot G, Sygusch J. Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus. Appl Environ Microbiol. 1990 Nov;56(11):3505-10.
Mandels,M et al. Problems and challenges in the cellulose to cellulase fermenation. Proc Biochem . 1978， 13;6-13
罗文新 夏宁邵 一种带增强子的原核高效表达载体的构建及初步应用 生物工程学报 ，2000，16卷5期，578-581
毕群 黄仪秀 朱圣庚 水蛭素基因的递归PCR合成和在大肠杆菌中的表达与分泌 北京大学学报（自然科学版），1997，第33卷，第6期
龙建银 王会信 刘莉 王芳 周廷冲 人转化生长因子β1在大肠杆菌中的分泌表达 中国生物化学与分子生物学学报，第15卷，第2期
袁涛 张雪梅 黄林 大肠杆菌分泌型表达重组人碱性成纤维细胞生长因子 华西药学杂志，1999，14（5-6），319-322
张同海 赵为诚 陈昆明 鲑鱼生长激素基因分泌型表达质粒的构建 生物化学与生物物理学进展，1994，21（4）
张宏权 王允玲 周廷冲 大肠杆菌分泌表达载体的构建和人表皮生长因子在大肠杆菌中的分泌性表达 生物化学杂志， 1995，第11卷第４期
黄仪秀 陈杰鹏 毕群 朱圣庚 应用PCR重组技术构建大肠杆菌分泌型表达载体 生物化学杂志，1995，第11卷，第6期
李军 李伯良 蛋白A信号肽引导的E.coli外泌高表达异源蛋白 生物化学与生物物理学报，1995，第27卷，第6期
宋礼华 桂向东 范清林 分泌型重组人生长激素的研究 生物学杂志，1999，Vol.16 No.1
邓建蓓 陈萍 王雨岭 带有蛋白标签的原核表达载体构建第四军医大学学报，1998，19（3）
刘永亮 童克忠 利用枯草杆菌碱性蛋白酶E基因的信号顺序构建分泌型表达载体 遗传学报 1994，21（3） 235-246
陈燕 李金照 夏令朝 人胰岛素原基因在大肠杆菌中高效外分泌表达 生物化学杂志 1995，Vol.11 No.6
蒙健宗 李晓明 王青艳 高密度培养基因工程大肠杆菌生产 α-乙酰乳酸脱羧酶 广西科学 2001，8（4）284-286，290
牛利国 陈志南 苏成芝 药立波抗 人肝癌单链抗体的基因克隆和分泌型表达细胞与分子免疫学杂志 1998，14（2） 102-109
刘纯杰 张兆山 李淑琴 黄翠芹幽门螺杆菌热休克蛋白A基因的克隆及高效分泌 表达军事医学科学院院刊 2000，9 Vol24 No.3 161-165
贺华君 杨卫东 施惠娟 吴祥甫 分泌癌胚抗原肿瘤靶向性Cu,Zn-SOD的基因克隆基因表达 华东理工大学学报 2001，4，Vol27 No.2 124-128
叶传忠 关明 张芳林 苏兵 分泌型肿瘤坏死因子相关激活诱导因子融合蛋白在大肠杆菌中的表达和纯化 复旦学报（医学科学版） 2001，7 28（4）
郭丽英 钟晓燕 吴祥甫 江苏蝮蛇神经生长因子在大肠杆菌中的表达及纯化生物工程学报 1999，7 Vol 15 No.3
梁爱华 苏智广 李晓玲 东亚钳蝎神经毒素在大肠杆菌中的表达 中国生物化学与分子生物学报 1999，4 Vo l5 No.2 205-210
赵宝华 许崇波 抗ａ型产气荚膜α菌毒素单链抗体基因的克隆和表达 生物工程学报 2001，9 Vo 17 No.5
江培诩 钱志康 黄伟达 大肠杆菌aroG基因在枯草杆菌中的分泌表达复旦学报(自然科学版) 2000,6 Vol 39 No.3
杨运桂 童芹 郑卫东 分子伴侣过量表达对蛋白质分泌及可溶性的影响 中国生物化学与分子生物学报 2000，6， Vol 16 No.3 382-387
盛涛 张建中 刘延清 利用自身ctxb启动子表达CTB 微生物免疫学进展 2001，Vol 29 No.4
韩照中 金明 冯尔玲 L-天门冬酰胺酶的可溶性表达研究 微生物学免疫学进展 2000，28（3）
王戈林 沈萍 杨澜 嗜麦芽假单胞菌酪氨酸酶基因在大肠杆菌中的克隆与表达 遗传学报 1999，26（3）274-279
陈建军 曹军卫 苏莉嗜酸嗜碱芽孢杆菌NTT76启动子和信号肽序列的克隆及表达 武汉大学学报理学版 2001，8 Vol 47 No.4 471-476
李武平 王志武 朱丽娜 张雪梅 刘丽军 分泌型重组人生长激素工程菌的高密度发酵 中国生物制品学杂志 2000，Vol 13 No.3、
唐雪明 邵蔚蓝 沈微 王正祥 枯草杆菌6816碱性蛋白酶基因在大肠杆菌中的克隆和表达 生物技术 2001，8 Vol 11 No.4
席全胜 钱欣国 周庆玮 潘巍人 epiregulin 在大肠杆菌中的表达 生物化学与生物物理学报 2000，32（3）295-298
张菊 颜真 韩苇 王俊楼 Angiostatin 基因的克隆及在原核细胞中的表达第四军医大学学报 1999，20（7） 607-612
安云庆 柯岩 杨贵贞 BPI23-Fcγ1重组蛋白原核表达的载体的构建、表达和生物学活性的测定 中国医学科学院学报 2000，Vol 22 No.6 511-516
林琳 任凤 赵宝昌 金晓艳 Balb/C小鼠金属硫蛋白基因-1在大肠杆菌中的高效表达 药物生物技术 2001，8（3）：139-142
王朝健 李元 CTLA-4在大肠杆菌中的克隆和表达 中国医学科学院学报 2001，4 Vol 23 No.2
李节 邱平铺，葡萄球菌蛋白A分泌型融合表达系统 国外医学、预防、诊断、治疗生物制品分册 1994，Vol 17 No.5 193-196
韩苇 颜真 王俊楼 赵永同 EPO模拟肽基因4串联体的构建和表达 第四军医大学学报 2001，22（4） 319-321
吴乃虎 基因工程原理 科学出版社 2001
卢圣栋 现代分子生物学实验技术（第二版） 中国协和医科大学出版社 1999
北京化学试剂公司 化学试剂手册 北京工业大学出版社 1993
方德福 周吕 丁濂 张德昌 现代医学实验技巧丛书 北京医科大学中国协和医科大学联合出版社 1996
彭秀玲 袁汉英 谢毅 王洪海基因工程实验技术 湖南科学技术出版社
朱玉贤 李毅 现代分子生物学 高等教育出版社 1998
周德庆 微生物学教程及实验方法 高等教育出版社
张龙翔 生物化学实验技术与方法 科学出版社
林万明 PCR实验技术指南 科学出版社
C.W.迪芬巴赫 G.S.德维克斯勒 著 黄培党 俞炜源 陈添弥 译 PCR技术实验指南 科学出版社 1998
邬敏辰 β-甘露聚糖酶及其水解产物的应用研究 江苏调味副食品 2001年(总第69期) 5-8
杨巍 张岚 卢智刚 翟中和 利用两种不相容质粒在大肠杆菌中共表达DEF45和DEF40 生物化学与生物物理学报 2001,33(2)：238-242
高宏斌 里氏木霉RUT—C30 β—甘露聚糖酶的诱导表达及其cDNA的克隆 内蒙古农业大学硕士学位论文 2003
范文斌 王龙 王和平 抗甘露聚糖酶多克隆抗体的制备及其ELISA双抗夹心法的建立 内蒙古农业大学学报 2004，24（4）89-93