抗人B7-2单链抗体基因在Tn5细胞、家蚕及哺乳动物细胞中的表达研究
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摘要
B7分子是一种重要的免疫细胞因子,属协同刺激分子(Costimulatory molecule)免疫球蛋白超家族(Immunoglobin superfamily, IgSF)成员,其中B7-2因其在APC上表达时间早、表达丰度高、易诱导表达,被认为是在免疫反应初期决定T细胞命运即对刺激应答或无应答的关键分子。近年来肿瘤免疫分子机理研究业已证实,抗B7单抗有望成为有效的治疗恶性肿瘤的生物制剂。另外,通过特异性阻断协同刺激信号B7/CD28以及CD40/CD40L的传导,可获得外周性免疫耐受。因此,对抗人B7-2单链抗体的表达和活性研究具有重要的应用价值。单链抗体由于分子小,减少了免疫原性,实体组织穿透力强,血浆半衰期短,因而在临床疾病的诊断和治疗中具有重要的应用价值。
本研究在成功制备抗人B7-2单链抗体的基础上取得了以下成果:
1.通过Bac to Bac杆状病毒表达系统,构建并筛选了含抗人B7-2单链抗体基因的重组AcBacmid和HyBacmid,并在昆虫细胞(Tn5)和BmN细胞-家蚕体内进行了表达;
2.构建了含信号肽的抗人B7-2单链抗体基因的哺乳动物细胞表达载体,并在哺乳动物细胞中进行了分泌表达;
3.对不同表达系统表达的抗人B7-2单链抗体进行了生物学活性的比较研究。
As a member of costimulatory molecule in immunoglobin superfamily, B7 is a important cytokine.Because being expressed early , abundantly and easy to be induced in APC, B7-2 is seen as the key element which decides if immune responses happen in the initial stage. In recent years, the molecular mechanism of tumor has been confirmed and anti-B7 antibody is expected to be an effective biological agents in treating malignant tumors. In addition, peripheral immune tolerance can be obtained by blocking the conduction of B7/CD28 and CD40/CD40L specifically. Therefore, the study in expression and activity of anti-human B7-2 single chain antibody has an important value. Single chain antibody in clinical diagnostic and therapeutic applications is valuable as they have been shown to have reduced immunogenicity, easy to penetrate into a solid tumor and have a shorter half life in plasma because of its smaller size.
In this research, we obtained the following results , based on the successful preparation of anti-human B7-2 single chain antibody:
1. AcBacmid and HyBacmid with the anti-human B7-2 single chain antibody was constructed and screened through the Bac-to-Bac baculovirus expression system. B7-2-ScFv was expressed in Tn5 cells , BmN cells and silkworms.
2. Mammalian cells’expression vector with the anti-human B7-2 single chain antibody was constructed and given a Secretory expression in mammalian cells.
3. Have a comparative study about the anti-human B7-2 single chain antibody with a signal peptide in different expression systems.
本研究在成功制备抗人B7-2单链抗体的基础上取得了以下成果:
1.通过Bac to Bac杆状病毒表达系统,构建并筛选了含抗人B7-2单链抗体基因的重组AcBacmid和HyBacmid,并在昆虫细胞(Tn5)和BmN细胞-家蚕体内进行了表达;
2.构建了含信号肽的抗人B7-2单链抗体基因的哺乳动物细胞表达载体,并在哺乳动物细胞中进行了分泌表达;
3.对不同表达系统表达的抗人B7-2单链抗体进行了生物学活性的比较研究。
As a member of costimulatory molecule in immunoglobin superfamily, B7 is a important cytokine.Because being expressed early , abundantly and easy to be induced in APC, B7-2 is seen as the key element which decides if immune responses happen in the initial stage. In recent years, the molecular mechanism of tumor has been confirmed and anti-B7 antibody is expected to be an effective biological agents in treating malignant tumors. In addition, peripheral immune tolerance can be obtained by blocking the conduction of B7/CD28 and CD40/CD40L specifically. Therefore, the study in expression and activity of anti-human B7-2 single chain antibody has an important value. Single chain antibody in clinical diagnostic and therapeutic applications is valuable as they have been shown to have reduced immunogenicity, easy to penetrate into a solid tumor and have a shorter half life in plasma because of its smaller size.
In this research, we obtained the following results , based on the successful preparation of anti-human B7-2 single chain antibody:
1. AcBacmid and HyBacmid with the anti-human B7-2 single chain antibody was constructed and screened through the Bac-to-Bac baculovirus expression system. B7-2-ScFv was expressed in Tn5 cells , BmN cells and silkworms.
2. Mammalian cells’expression vector with the anti-human B7-2 single chain antibody was constructed and given a Secretory expression in mammalian cells.
3. Have a comparative study about the anti-human B7-2 single chain antibody with a signal peptide in different expression systems.
引文
[1]王廷华,李官成, Xin-Fu Zhou,等.抗体理论与技术[M],北京:科学出版社, 2009, 8–9.
[2] Brekke O, Sandl I I. Therapeutic antibodies for human diseases at the dawn of the twenty-first century [J]. N at Rev D rug D iscov, 2003, 2(1): 52-62.
[3]胡春艳,刘全忠.单克隆抗体制备技术及应用的研究进展[J].安徽预防医学杂志, 2008, 14(2) : 116 - 118.
[4]沈倍奋.治疗性抗体的改造[J].医学分子生物学杂志, 2006,3(4): 245 - 249.
[5]费晋劳,任军,苗继廷.单链抗体构建、表达及临床应用的研究现状[J].陕西医学杂志, 2000, 29(1): 30-32.
[6] Holliger P, Prospero T, Winter G. "Diabody": Small bivalent and bispecific antibody fragments [J]. Proc Natl Acad Sci USA, 1993, 90(14):6444-6448.
[7]杨丽娟,侯颖春,白玉杰,等.抗胃癌单链抗体可变区序列分析及空间结构模拟[J].世界华人消化杂志,2008,16 (21):2333-2336.
[8]张宏斌,江悦华,王捷,等.抗人膀胱癌噬菌体单链抗体的初步制备[J].免疫学杂志,1997,13(2):119-121.
[9]钟小林,高会广,吉清,等.一种可溶性单抗ScFv片段的表达及鉴定[J].第三军医大学学报, 2003, 25(8): 691-694.
[10] Zitzmann S, Mier W, Schad A, et al. A new prostate carcinoma binding peptide (DUP21) for tumor imaging and therapy [J]. Clin Cancer Res, 2005, 11(1): 139-146.
[11]段红,李少林.单链抗体及其在肿瘤免疫上的应用[J].国际肿瘤学杂志, 2006, 33(7) : 487-489.
[12]昌晓红,崔恒,冯捷.卵巢癌抗独特型微抗体疫苗诱导BALB/c小鼠体内抗肿瘤免疫应答的实验研究[J].癌症, 2004, 23 (7): 777-781.
[13] Naoki S, Toshio K, Masanori S, et al. SEA-ScFv as a bifunctional antibody: construction of a bacterial expression system and it s functional analysis[J]. Biochem Biophys Res Communica, 1999, 256:223-230.
[14] Wiseman GA, Leigh B, Erwin WD, et al. Radiation dosimetry results for Zevalin radio immunotherapy of rituximab-refractory nonHodgkin lymphoma [J]. Cancer, 2002, 94(4) Suppl: 1349-1357.
[15] Skrepnik N, Zieske AW, Robert E, et al. Aggressive administration of recombinant oncotoxin AR209 (anti ErbB-2) in athymic nude mice implanted with orthotopic human non-small cell lung tumours [J]. Cancer, 1998, 34(10):1628 -1633.
[16] Skrepnik EN, Zieske AW, Bravo JC, et al. Recombinantoncotoxin AR209 (anti P185 erbB-2) diminishes human prostate carcinoma xenografts [J]. Urol, 1999, 161(7):984-989.
[17] Azemar M, Schmidt M, Arlt F, et al. Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapid tumor regression in vivo[J]. Cancer, 2000, 86(2):269-275.
[18] Azemar M, Djahansouzi S, Jagr E, et al. Regression of cutaneous tumor lesions in patients intratumorally injected with a recombinant single-chain antibody-toxin targeted to ErbB2/HER2[J]. Breast Cancer Res Treat, 2003, 82(3):155-164.
[19] Noronha EJ, Wang X, Desai SA, et al. Limited diversity of human scFv fragmentsisolated by panning a synthetic phage-display scFv library with cultured human melanoma cells [J]. Immunol, 1998, 161(6):2968-2976.
[20] Topping KP, Hough VC, Monson JR, et al. Isolation of human colorectal tumor reactive antibodies using phage display technology [J]. Int Oncol, 2000, 16(1):187-195.
[21] Ridgway JB, Ng E, Kern JA, et al. Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumour and nontumor cell lines [J]. Cancer Res, 1999, 59(11):2718-2723.
[22] Guo JQ, Li QM,Zhou JY, et al. Efficient recovery of the functional IP10-scFv fusion portein from inclusion bodies with an no-column refoding system [J]. Protein Expr Purif, 2006, 45(1):168-174.
[23] Krauss J, Arn MA, Vu BK, et al. Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein [J]. Biochem Biophys Res Commun2005, 331(2):595-602.
[24]白文涛,徐志凯,张芳琳,等.抗汉坦病毒NP抗原细胞内抗体的瞬时表达及活性检测[J].细胞与分子免疫学杂志,2004, 20 (6): 705-707.
[25]邵锴,姜海燕,张国利,等.从噬菌体抗体库中筛选抗HIV2 Tat蛋白单链抗体[J].中国实验诊断学, 2008, 12 (5): 592-594.
[26]顾长国,刘琛,李磊.人源化抗HCMV噬菌体ScFv库的构建[J].免疫学杂志, 2004, 20(6): 468-471.
[27]司炳银,杨佩英,秦鄂德,等.抗登革3型病毒单链抗体的可溶性表达和鉴定[J].中华实验和临床病毒学杂志, 1998, 12 (4): 350-352.
[28]何卓,汪世平,肖小芹,等.日本血吸虫未成熟卵单链抗体库的构建、筛选及初步应用[J].生物化学与生物物理进展, 2008, 35(8): 921-928.
[29] White GP, Meeusen EN, Newton SE. A single chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gast rointestinal nematode parasite Haemonchus contortus [J]. Vet Immunol Immunopathol, 2001, 78(2): 117-129.
[30] Yoshida S ,Ioka D ,Mat suoka H ,et al . Bacteriaex pressing single chain immunotoxin inhibit malaria parasite development in mosquitoes[J]. Mol BiochemParasitol, 2001, 113(1): 89-96.
[31] Hagemeyer CE , Tomic I ,J aminet P ,et al . Fibrin-targeted direct factor Xa inhibition :construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single chain antibody and tick anticoagulant peptide [J]. Thromb Haemost, 2004, 92(1): 47-53.
[32] Rit sch A , Ebenbichler C ,Naschberger E ,et al . Phage displayed recombinant single chain antibody fragments with high affinity for cholesteryl ester transfer protein (CETP) :cDNA cloning , characterization and CETP quantification [J]. Clin Chem Lab Med, 2004, 42(3): 247-255.
[33] Russell J S, Raju U , Gumin GJ , et al . Inhibition of radiation-induced nuclear factor-κB activation by an anti-Ras single-chain antibody fragment [J]. Cancer Res, 2002, 62(8): 2318-2326.
[34] Fengfeng Z, Yuhua Q, Jiang Z, et al . Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli [J]. Cytotechnology, 2009, 60: 85-94.
[35] Van Regenmortel MHV , Fauquet CM,Bishop DHL , et al . Seventh report of the international ommittee on taxonomy of viruses [J]. Virus Taxonomy, 2000 (4): 999 - 1002.
[36] Martin BM, Carbone E, Yatani A, et al . Amino acid sequence and physiological characterization of toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann [J]. Toxicon, 1988,26(9):785-794.
[37] Merrington C L. Baculovirus expression systems. In“Protein Expression: a practical approach”(M). New York: Oxford University press Inc, 1999. 101-127.
[38]吕鸿声.昆虫病毒分子生物学[M].北京:中国农业科技出版社, 1998.
[39]黄光镁,朱江等,抗人CD28单链抗体基因在Sf9细胞及家蚕中的表达研究,苏州大学, 2009, 09.
[40] Gandor C , Zang-Gandor M , Flor P , et al. Conditionally adherent growth of serum—independent CHO cells for automated drug screening and biopharmaceutical production [J]. Biotechnol Biocng, 1999, 65: 523-528.
[41] Wittel UA , J ain M , Goel A , et al . The in vivo characteristics of geneticallyengineered divalent and tet ravalent single2chain antibody constructs [J]. Nucl Med Biol, 2005, 32(2): 157-64.
[42] Shen S, Forero A , LoBuglio AF , et al . Patient-specific dosimetry of pretargeted radioimmunot herapy using CC49 fusion protein in patients with gast rointestinal malignancies [J]. J Nucl Med , 2005,46(4):642-651
[43] Kenanova V, Olaf sen T, Williams L E, et al. Radioiodinated versus radiometal-labeled anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments: optimal pharmacokinetics for therapy [J] .Cancer Res, 2007, 67(2):718-726.
[44] Ali M , Suzuki H , Fukuba T , et al . Improvements in the cell-free production of functional antibodies using cell extract from protease deficient Escherichia coli mutant [J]. J Biosci Bioeng, 2005, 99(2):181-186.
[45] Pujol M , Ramirez NI , Ayala M , et al . An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification [J] . Vaccine, 2005, 23(15):1833-1837.
[1] Luckow V A , Lee S C , barry G F , Olins P O. Efficient generation of insectious recombinant baculorviruses by site-specific transposon mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli [J]. Virol , 1993 ,67: 4566-4579.
[2] Wu X F , Cao C P , Xu Y X , et al. Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV, and knockout of cysteinase gene for more efficient expression[J]. Science in China ( Ser. C Life Sciences),2004,47(5): 406-415
[3] Lu Hongsheng. Molecular Biology of Insect Viruses , China Agricultueal Science and Technology Press ,1998 : 554
[4]孙传洋,单玉喜,胡玉敏,等.抗B7-1和抗CD40L单抗延长小鼠皮肤移植物存活实验研究[J].现代免疫学, 2005, 25 (4): 319-322
[5]郑峰丰,陈永井,朱江,等.抗人CD-28单链抗体基因及其昆虫杆状病毒重组转移载体的构建[J].中国基础与临床医学杂志, 2006, 5(1): 3-6
[6] Fengfeng Zheng, Yuhua Qiu, Jiang Zhu et al. Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli [J]. Cytoechnology, 2009, 60: 85-94
[7] Grosse Hovest L, Hartlapp I, Marwan W, et al. A recombinant bispecific single chain antibody induces targeted, supra-agonistic CD28 stimulation and tumor cellkilling[J]. Eur J Immunol.2003, 33(5): 334-340.
[8] Nieroda CA, Milenic DE, Carrasquillo JA, et al. Improved tumor radioimmuno-detection using a single chain Fv and gamma-interferon: Potential clinical applications for radioimmuno guided surgery and gamma scanning[J]. Cancer Res. 1995, 55(13): 285-288.
[9] Kiefhaber T, Rudolph R,Kohler H-H et al. Protein aggregation in vitro and in vivo:a quantitative model of the kinetic competition between folding and aggregation[J]. Biol Technology, 1991, 9: 825-829
[10] Yeh Sih, Rousseau D. Folding intermediates in cytochrome c [J]. Natu Stru Biol, 1998, 5(3): 222
[11]刘国奇,王海涛.外源蛋白在中国仓鼠卵母细胞中的高效表达的策略[J].生物化学与生物物理进展, 2000, 27(5): 496
[2] Brekke O, Sandl I I. Therapeutic antibodies for human diseases at the dawn of the twenty-first century [J]. N at Rev D rug D iscov, 2003, 2(1): 52-62.
[3]胡春艳,刘全忠.单克隆抗体制备技术及应用的研究进展[J].安徽预防医学杂志, 2008, 14(2) : 116 - 118.
[4]沈倍奋.治疗性抗体的改造[J].医学分子生物学杂志, 2006,3(4): 245 - 249.
[5]费晋劳,任军,苗继廷.单链抗体构建、表达及临床应用的研究现状[J].陕西医学杂志, 2000, 29(1): 30-32.
[6] Holliger P, Prospero T, Winter G. "Diabody": Small bivalent and bispecific antibody fragments [J]. Proc Natl Acad Sci USA, 1993, 90(14):6444-6448.
[7]杨丽娟,侯颖春,白玉杰,等.抗胃癌单链抗体可变区序列分析及空间结构模拟[J].世界华人消化杂志,2008,16 (21):2333-2336.
[8]张宏斌,江悦华,王捷,等.抗人膀胱癌噬菌体单链抗体的初步制备[J].免疫学杂志,1997,13(2):119-121.
[9]钟小林,高会广,吉清,等.一种可溶性单抗ScFv片段的表达及鉴定[J].第三军医大学学报, 2003, 25(8): 691-694.
[10] Zitzmann S, Mier W, Schad A, et al. A new prostate carcinoma binding peptide (DUP21) for tumor imaging and therapy [J]. Clin Cancer Res, 2005, 11(1): 139-146.
[11]段红,李少林.单链抗体及其在肿瘤免疫上的应用[J].国际肿瘤学杂志, 2006, 33(7) : 487-489.
[12]昌晓红,崔恒,冯捷.卵巢癌抗独特型微抗体疫苗诱导BALB/c小鼠体内抗肿瘤免疫应答的实验研究[J].癌症, 2004, 23 (7): 777-781.
[13] Naoki S, Toshio K, Masanori S, et al. SEA-ScFv as a bifunctional antibody: construction of a bacterial expression system and it s functional analysis[J]. Biochem Biophys Res Communica, 1999, 256:223-230.
[14] Wiseman GA, Leigh B, Erwin WD, et al. Radiation dosimetry results for Zevalin radio immunotherapy of rituximab-refractory nonHodgkin lymphoma [J]. Cancer, 2002, 94(4) Suppl: 1349-1357.
[15] Skrepnik N, Zieske AW, Robert E, et al. Aggressive administration of recombinant oncotoxin AR209 (anti ErbB-2) in athymic nude mice implanted with orthotopic human non-small cell lung tumours [J]. Cancer, 1998, 34(10):1628 -1633.
[16] Skrepnik EN, Zieske AW, Bravo JC, et al. Recombinantoncotoxin AR209 (anti P185 erbB-2) diminishes human prostate carcinoma xenografts [J]. Urol, 1999, 161(7):984-989.
[17] Azemar M, Schmidt M, Arlt F, et al. Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapid tumor regression in vivo[J]. Cancer, 2000, 86(2):269-275.
[18] Azemar M, Djahansouzi S, Jagr E, et al. Regression of cutaneous tumor lesions in patients intratumorally injected with a recombinant single-chain antibody-toxin targeted to ErbB2/HER2[J]. Breast Cancer Res Treat, 2003, 82(3):155-164.
[19] Noronha EJ, Wang X, Desai SA, et al. Limited diversity of human scFv fragmentsisolated by panning a synthetic phage-display scFv library with cultured human melanoma cells [J]. Immunol, 1998, 161(6):2968-2976.
[20] Topping KP, Hough VC, Monson JR, et al. Isolation of human colorectal tumor reactive antibodies using phage display technology [J]. Int Oncol, 2000, 16(1):187-195.
[21] Ridgway JB, Ng E, Kern JA, et al. Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumour and nontumor cell lines [J]. Cancer Res, 1999, 59(11):2718-2723.
[22] Guo JQ, Li QM,Zhou JY, et al. Efficient recovery of the functional IP10-scFv fusion portein from inclusion bodies with an no-column refoding system [J]. Protein Expr Purif, 2006, 45(1):168-174.
[23] Krauss J, Arn MA, Vu BK, et al. Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein [J]. Biochem Biophys Res Commun2005, 331(2):595-602.
[24]白文涛,徐志凯,张芳琳,等.抗汉坦病毒NP抗原细胞内抗体的瞬时表达及活性检测[J].细胞与分子免疫学杂志,2004, 20 (6): 705-707.
[25]邵锴,姜海燕,张国利,等.从噬菌体抗体库中筛选抗HIV2 Tat蛋白单链抗体[J].中国实验诊断学, 2008, 12 (5): 592-594.
[26]顾长国,刘琛,李磊.人源化抗HCMV噬菌体ScFv库的构建[J].免疫学杂志, 2004, 20(6): 468-471.
[27]司炳银,杨佩英,秦鄂德,等.抗登革3型病毒单链抗体的可溶性表达和鉴定[J].中华实验和临床病毒学杂志, 1998, 12 (4): 350-352.
[28]何卓,汪世平,肖小芹,等.日本血吸虫未成熟卵单链抗体库的构建、筛选及初步应用[J].生物化学与生物物理进展, 2008, 35(8): 921-928.
[29] White GP, Meeusen EN, Newton SE. A single chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gast rointestinal nematode parasite Haemonchus contortus [J]. Vet Immunol Immunopathol, 2001, 78(2): 117-129.
[30] Yoshida S ,Ioka D ,Mat suoka H ,et al . Bacteriaex pressing single chain immunotoxin inhibit malaria parasite development in mosquitoes[J]. Mol BiochemParasitol, 2001, 113(1): 89-96.
[31] Hagemeyer CE , Tomic I ,J aminet P ,et al . Fibrin-targeted direct factor Xa inhibition :construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single chain antibody and tick anticoagulant peptide [J]. Thromb Haemost, 2004, 92(1): 47-53.
[32] Rit sch A , Ebenbichler C ,Naschberger E ,et al . Phage displayed recombinant single chain antibody fragments with high affinity for cholesteryl ester transfer protein (CETP) :cDNA cloning , characterization and CETP quantification [J]. Clin Chem Lab Med, 2004, 42(3): 247-255.
[33] Russell J S, Raju U , Gumin GJ , et al . Inhibition of radiation-induced nuclear factor-κB activation by an anti-Ras single-chain antibody fragment [J]. Cancer Res, 2002, 62(8): 2318-2326.
[34] Fengfeng Z, Yuhua Q, Jiang Z, et al . Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli [J]. Cytotechnology, 2009, 60: 85-94.
[35] Van Regenmortel MHV , Fauquet CM,Bishop DHL , et al . Seventh report of the international ommittee on taxonomy of viruses [J]. Virus Taxonomy, 2000 (4): 999 - 1002.
[36] Martin BM, Carbone E, Yatani A, et al . Amino acid sequence and physiological characterization of toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann [J]. Toxicon, 1988,26(9):785-794.
[37] Merrington C L. Baculovirus expression systems. In“Protein Expression: a practical approach”(M). New York: Oxford University press Inc, 1999. 101-127.
[38]吕鸿声.昆虫病毒分子生物学[M].北京:中国农业科技出版社, 1998.
[39]黄光镁,朱江等,抗人CD28单链抗体基因在Sf9细胞及家蚕中的表达研究,苏州大学, 2009, 09.
[40] Gandor C , Zang-Gandor M , Flor P , et al. Conditionally adherent growth of serum—independent CHO cells for automated drug screening and biopharmaceutical production [J]. Biotechnol Biocng, 1999, 65: 523-528.
[41] Wittel UA , J ain M , Goel A , et al . The in vivo characteristics of geneticallyengineered divalent and tet ravalent single2chain antibody constructs [J]. Nucl Med Biol, 2005, 32(2): 157-64.
[42] Shen S, Forero A , LoBuglio AF , et al . Patient-specific dosimetry of pretargeted radioimmunot herapy using CC49 fusion protein in patients with gast rointestinal malignancies [J]. J Nucl Med , 2005,46(4):642-651
[43] Kenanova V, Olaf sen T, Williams L E, et al. Radioiodinated versus radiometal-labeled anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments: optimal pharmacokinetics for therapy [J] .Cancer Res, 2007, 67(2):718-726.
[44] Ali M , Suzuki H , Fukuba T , et al . Improvements in the cell-free production of functional antibodies using cell extract from protease deficient Escherichia coli mutant [J]. J Biosci Bioeng, 2005, 99(2):181-186.
[45] Pujol M , Ramirez NI , Ayala M , et al . An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification [J] . Vaccine, 2005, 23(15):1833-1837.
[1] Luckow V A , Lee S C , barry G F , Olins P O. Efficient generation of insectious recombinant baculorviruses by site-specific transposon mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli [J]. Virol , 1993 ,67: 4566-4579.
[2] Wu X F , Cao C P , Xu Y X , et al. Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV, and knockout of cysteinase gene for more efficient expression[J]. Science in China ( Ser. C Life Sciences),2004,47(5): 406-415
[3] Lu Hongsheng. Molecular Biology of Insect Viruses , China Agricultueal Science and Technology Press ,1998 : 554
[4]孙传洋,单玉喜,胡玉敏,等.抗B7-1和抗CD40L单抗延长小鼠皮肤移植物存活实验研究[J].现代免疫学, 2005, 25 (4): 319-322
[5]郑峰丰,陈永井,朱江,等.抗人CD-28单链抗体基因及其昆虫杆状病毒重组转移载体的构建[J].中国基础与临床医学杂志, 2006, 5(1): 3-6
[6] Fengfeng Zheng, Yuhua Qiu, Jiang Zhu et al. Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli [J]. Cytoechnology, 2009, 60: 85-94
[7] Grosse Hovest L, Hartlapp I, Marwan W, et al. A recombinant bispecific single chain antibody induces targeted, supra-agonistic CD28 stimulation and tumor cellkilling[J]. Eur J Immunol.2003, 33(5): 334-340.
[8] Nieroda CA, Milenic DE, Carrasquillo JA, et al. Improved tumor radioimmuno-detection using a single chain Fv and gamma-interferon: Potential clinical applications for radioimmuno guided surgery and gamma scanning[J]. Cancer Res. 1995, 55(13): 285-288.
[9] Kiefhaber T, Rudolph R,Kohler H-H et al. Protein aggregation in vitro and in vivo:a quantitative model of the kinetic competition between folding and aggregation[J]. Biol Technology, 1991, 9: 825-829
[10] Yeh Sih, Rousseau D. Folding intermediates in cytochrome c [J]. Natu Stru Biol, 1998, 5(3): 222
[11]刘国奇,王海涛.外源蛋白在中国仓鼠卵母细胞中的高效表达的策略[J].生物化学与生物物理进展, 2000, 27(5): 496