α-N-乙酰半乳糖胺酶突变文库的建立及筛选
|
摘要
α-N-乙酰半乳糖胺酶是血型转换的工具酶,可以降解A型红细胞表面抗原、消除其抗原抗体反应,在输血治疗中,可以避免血型配型带来的不便。本实验室从报道的α-N-乙酰半乳糖胺酶中选取活性最好的酶,构建α-N-乙酰半乳糖胺酶的原核表达载体,表达并纯化α-N-乙酰半乳糖胺酶,检测酶活性。利用定点突变技术,在关键位点进行突变,用高通量筛选技术筛选突变文库,期望得到较高活性的α-N-乙酰半乳糖胺酶。本课题的研究工作主要包括以下两部分：
1.α-N-乙酰半乳糖胺酶的构建
为了建立新型α-N-乙酰半乳糖胺酶的筛选、检测方法,我们提取脑膜金黄杆菌的基因组DNA,以此为模板PCR扩增出α-N-乙酰半乳糖胺酶(A4)。将A4克隆至pET-24a载体,转化B121表达菌株进行蛋白表达。使用亲和层析方法纯化His-A4酶,选择显色底物验证酶活性。同时,我们改进了传统的ELISA方法,直接将红细胞膜包被于ELISA检测平板中,以红细胞膜表面抗原作为直接底物,用ELISA方法检测酶活性。此研究建立了新型ELISA实验方法,以此方法验证了A4酶的活性,证明了此酶能够有效降低红细胞表面抗原抗体反应,且具有浓度和时间依赖性。
2.α-N-乙酰半乳糖胺酶突变文库的筛选
自然界中的α-N-乙酰半乳糖胺酶活性较低,不足以大规模生产酶用于通用型血的制备。在本实验中,使用定点突变技术结合高通量筛选方法,期望得到活性较高的糖苷酶。我们突变了酶的6个氨基酸位点分别是96、98、179、181、225、228,筛选了2400个左右的重组子,从实验中可知,179、181、225、228位置突变后酶的活性大幅度下降,因此这四个位点对酶的活性至关重要。96、98这两个位点突变后,有4个突变酶活性相当,它们突变后活性改变的机理需要进一步研究。
Alpha-a-N-acetylgalactosaminidase belongs to an important family of enzymes that can be used to degrade surface antigen on the surface of A-type red blood cells to eliminate the antigen-antibody reaction. It can help to relieves difficulties associate with typing and crossmatching in blood transfusion. The best activity of a-N-acetylgalactosaminidase was selected and the recombinant plasmid was constructed. With the expression and purification of a-N- acetylgalactosaminidase, its enzyme kinetics were measured. The key site of the enzyme was mutated by PCR directed mutagenesis. We hoped to get the higher efficiency of a-N-acetylgalactosaminidase through high-throughput screening. The main objectives are as follows:
1. The construction ofa-N-acetylgalactosaminidase
The coding region of an a-N-acetylgalactosaminidase (A4) was PCR amplified from genomic DNA of Chryseobacterium meningosepticum and subcloned into pET24a-A4 for overexpression His-A4. The overexpressed His-A4 enzyme was purified using affinity chromatography and has activity that is comparable with literature reported using a conventional method with an artificial substrate. To better measure the activity of a-N-acetylgalactosaminidase in real application, we established a novel method that directly use the surface antigen of red blood cell as substrate and use ELISA to detect the un-cleaved antigen. The activity of His-A4 was evaluated in the new ELISA method and was demonstrated to be able to decrease the blood cell surface antigen-antibody reaction in concentration- and time-dependent fashion.
2. The screening of a-N-acetylgalactosaminidase mutation libraries
The major obstacle to use glycosidase in the preparation of universal blood is their low enzymatic activity. We hoped to get the higher efficiency enzyme by directed mutagenesis and high-throughput screening. The site of amino acid were 96,98,179,181,225 and 228 separately. We screened about 2,400 recombinant bacteria. The results showed that 179,181,225 and 228 these four sites are the key sites for the enzyme because the activity of a-N-acetylgalactosaminidase has been lost after the mutation. We got the four recombinant bacterias. Their activities were comparable to the wild bacteria. The mechanism of the changing enzyme activity should be further studied.
1.α-N-乙酰半乳糖胺酶的构建
为了建立新型α-N-乙酰半乳糖胺酶的筛选、检测方法,我们提取脑膜金黄杆菌的基因组DNA,以此为模板PCR扩增出α-N-乙酰半乳糖胺酶(A4)。将A4克隆至pET-24a载体,转化B121表达菌株进行蛋白表达。使用亲和层析方法纯化His-A4酶,选择显色底物验证酶活性。同时,我们改进了传统的ELISA方法,直接将红细胞膜包被于ELISA检测平板中,以红细胞膜表面抗原作为直接底物,用ELISA方法检测酶活性。此研究建立了新型ELISA实验方法,以此方法验证了A4酶的活性,证明了此酶能够有效降低红细胞表面抗原抗体反应,且具有浓度和时间依赖性。
2.α-N-乙酰半乳糖胺酶突变文库的筛选
自然界中的α-N-乙酰半乳糖胺酶活性较低,不足以大规模生产酶用于通用型血的制备。在本实验中,使用定点突变技术结合高通量筛选方法,期望得到活性较高的糖苷酶。我们突变了酶的6个氨基酸位点分别是96、98、179、181、225、228,筛选了2400个左右的重组子,从实验中可知,179、181、225、228位置突变后酶的活性大幅度下降,因此这四个位点对酶的活性至关重要。96、98这两个位点突变后,有4个突变酶活性相当,它们突变后活性改变的机理需要进一步研究。
Alpha-a-N-acetylgalactosaminidase belongs to an important family of enzymes that can be used to degrade surface antigen on the surface of A-type red blood cells to eliminate the antigen-antibody reaction. It can help to relieves difficulties associate with typing and crossmatching in blood transfusion. The best activity of a-N-acetylgalactosaminidase was selected and the recombinant plasmid was constructed. With the expression and purification of a-N- acetylgalactosaminidase, its enzyme kinetics were measured. The key site of the enzyme was mutated by PCR directed mutagenesis. We hoped to get the higher efficiency of a-N-acetylgalactosaminidase through high-throughput screening. The main objectives are as follows:
1. The construction ofa-N-acetylgalactosaminidase
The coding region of an a-N-acetylgalactosaminidase (A4) was PCR amplified from genomic DNA of Chryseobacterium meningosepticum and subcloned into pET24a-A4 for overexpression His-A4. The overexpressed His-A4 enzyme was purified using affinity chromatography and has activity that is comparable with literature reported using a conventional method with an artificial substrate. To better measure the activity of a-N-acetylgalactosaminidase in real application, we established a novel method that directly use the surface antigen of red blood cell as substrate and use ELISA to detect the un-cleaved antigen. The activity of His-A4 was evaluated in the new ELISA method and was demonstrated to be able to decrease the blood cell surface antigen-antibody reaction in concentration- and time-dependent fashion.
2. The screening of a-N-acetylgalactosaminidase mutation libraries
The major obstacle to use glycosidase in the preparation of universal blood is their low enzymatic activity. We hoped to get the higher efficiency enzyme by directed mutagenesis and high-throughput screening. The site of amino acid were 96,98,179,181,225 and 228 separately. We screened about 2,400 recombinant bacteria. The results showed that 179,181,225 and 228 these four sites are the key sites for the enzyme because the activity of a-N-acetylgalactosaminidase has been lost after the mutation. We got the four recombinant bacterias. Their activities were comparable to the wild bacteria. The mechanism of the changing enzyme activity should be further studied.
引文
1. Landsteiner, K., Agglutination phenomena of normal human blood. Wien Klin Wochenschr,2001.113(20-21):p.768-9.
2. Watkins, W.M., Biochemistry and Genetics of the ABO, Lewis, and P blood group systems. Adv Hum Genet,1980.10:p.1-136,379-85.
3. 章扬培,季守平,杨军,血型转变.中国实验血液学杂志,1998.6(2)：p.91-97.
4. Liu, Q.P., et al., Bacterial glycosidases for the production of universal red blood cells. Nat Biotechnol,2007.25(4):p.454-64.
5. Baeher, R.V. Biotechnology of blood. Biotechnology,1991.19:p.1-463.
6. Goldstein, J., et al., Group B erythrocytes enzymatically converted to group O survive normally in A, B, and O individuals. Science,1982.215(4529):p.168-70.
7. Kruskall, M.S., et al., Transfusion to blood group A and O patients of group B RBCs that have been enzymatically converted to group O. Transfusion,2000.40(11):p. 1290-8.
8. Zhu, A. and J. Goldstein, Cloning and functional expression of a cDNA encoding coffee bean alpha-galactosidase. Gene,1994.140(2):p.227-31.
9. Olsson, M.L. and H. Clausen, Modifying the red cell surface:towards an ABO-universal blood supply. Br J Haematol,2008.140(1):p.3-12.
10. Daniels, G and S.G. Withers, Towards universal red blood cells. Nat Biotechnol,2007. 25(4):p.427-8.
11. Lenny, L.L. and J. Goldstein, The production of group O cells. Biotechnology,1991. 19:p.75-100.
12. LaVecchio, J.A., A.D. Dunne, and A.S. Edge, Enzymatic removal of alpha-galactosyl epitopes from porcine endothelial cells diminishes the cytotoxic effect of natural antibodies. Transplantation,1995.60(8):p.841-7.
13. 杨军,宫锋,季守平,B→O血型转变工具酶α-半乳糖苷酶cDNA克隆及表达.中国生物化学与分子生物学报,2000.16(4)：p.438-442.
14. Stemmer, W.P., Rapid evolution of a protein in vitro by DNA shuffling. Nature,1994. 370(6488):p.389-91.
15. Harayama, S., Artificial evolution by DNA shuffling. Trends Biotechnol,1998.16(2):p. 76-82.
16. Chen, K. and F.H. Arnold, Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci U S A,1993.90(12):p.5618-22.
17. 曾家豫,杨国兵,廖世奇,生物酶的定向进化及其应用.卫生职业教育,2007.25(23)：p.155-159.
18. Kone, F.M., et al., Digital screening methodology for the directed evolution of transglycosidases. Protein Eng Des Sel,2009.22(1):p.37-44.
19. Ben-David, A., G. Shoham, and Y. Shoham, A universal screening assay for glycosynthases:directed evolution of glycosynthase XynB2(E335G) suggests a general path to enhance activity. Chem Biol,2008.15(6):p.546-51.
20. Landsteiner, K., Zur Kenntnis der antifermentativen, lytischen und agglutinierenden Wirkungen des Blutserums und der Lymphe. Zentralblatt Bakteriologie 1900(27):p. 357-362.
21. Donald, A.S., et al., The peptide moiety of human-blood-group active glycoproteins associated with the ABO and Lewis groups. Biochem J,1969.115(1):p.125-7.
22. Morgan, W.T. and W.M. Watkins, Genetic and biochemical aspects of human blood-group A-, B-, H-, Le-a- and Le-b-specificity. Br Med Bull,1969.25(1):p.30-4.
23. Morgan, W.T. and W.M. Watkins, Some aspects of the biochemistry of the human blood-group substances. Br Med Bull,1959.15(2):p.109-13.
24. Scott, M.D., et al., Chemical camouflage of antigenic determinants:stealth erythrocytes. Proc Natl Acad Sci U S A,1997.94(14):p.7566-71.
25. 张印则,红细胞抗原修饰的化学进展.中国输血杂志,2003.16(6)：p.435-437.
26. Abuchowski, A., et al., Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J Biol Chem,1977.252(11):p. 3578-81.
27. Fuertges F, A.A., The clinical efficacy of poly(ethyleneglycol) modified proteins. Control Release,1990(11):p.139-148.
28. Macchiarini, P., et al., Evidence of human non-alpha-galactosyl antibodies involved in the hyper acute rejection of pig lungs and their removal by pig organ perfusion. J Thorac Cardiovasc Surg,1998.116(5):p.831-43.
29. 季守平,刘泽澎,任会明,甲氧基聚乙二醇遮蔽红细胞表面抗原制备通用型血液的初步研究.中国输血杂志,2000.13(4)：p.223-227.
30. Hortin, G.L., H.T. Lok, and S.T. Huang, Progress toward preparation of universal donor red cells. Artif Cells Blood Substit Immobil Biotechnol,1997.25(5):p.487-91.
31. 檀英霞,王捷熙,章扬培,体外化学修饰红细胞——通用型血?.生命科学,2004.16(1).
32. 赵瑞东,王增祺,魏秀萍,红细胞ABO血型抗原表达及化学修饰.广州医药.38(4)：p.6-9.
33. 殷文劫,阮长耿,红细胞血型抗原修饰的研究与展望.苏州大学学报,2007.27(1)：p.68-72.
34. 季守平,刘泽澎,任会明,甲氧基聚乙二醇遮蔽红细胞表面抗原制备通用型血液的初步研究.中国输血杂志,2000.13(4)：p.223-227.
35. Lenny, L.L., et al., Multiple-unit and second transfusions of red cells enzymatically converted from group B to group O:report on the end of phase 1 trials. Transfusion, 1995.35(11):p.899-902.
36. Lenny, L.L., et al., Transfusions to group O subjects of 2 units of red cells enzymatically converted from group B to group O. Transfusion,1994.34(3):p. 209-14.
37. Zhu, A., et al., Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes. Arch Biochem Biophys,1996.327(2):p.324-9.
38.血型转换器.新技术：p.21.
39. 章扬培,杨军,高新,用基因重组α-半乳糖苷酶进行B→O血型改造.科学通报,2003.48(1)：p.43-47.
40. 高红伟,高新,鲍国强,基因重组α-半乳糖苷酶的制备工艺研究.生物技术通讯,2004.15(6)：p.566-569.
41. 鲍国强,高红伟,宫锋,基因重组α-半乳糖苷酶的生化性质研究.军事医学科学院院刊,2005.29(2)：p.139-141.
42. 宫峰,吕秋霜,由英,用α-半乳糖苷酶制备可供输注的人通用型O型红细胞.中国实验血液学杂志,2005.29(2)：p.139-141.
43. 刘泽澎,张成林,卢雁平,α-半乳糖苷酶酶解B型长臂猿红细胞回输A型长臂猿的研究.中国生物化学和分子生物学报,2004.20(2)：p.229-233.
44. 郁成雨,徐华,王立生,张建耕,章扬培,新型高比活力α-N-乙酰半乳糖胺酶应用于人红细胞.科学通报,2008.53(11)：p.1288-1295.
45. 徐华,郁成雨,章扬培,联合应用α-半乳糖苷酶和α-N-乙酰半乳糖胺酶实现红细胞AB→O血型转变.中国输血杂志,2008.21(12)：p.917-920.
46. 谢晚彬,谢和芳,蛋白质定向进化的研究技术及应用.中国生物工程杂志,2005：p.16-18.
47. 方柏山,郑媛媛,酶体外定向进化[I]——突变基因文库构建技术及其新进展.华侨大学学报,2004.25(4)：p.337-342.
48. 徐威,朱春宝,朱宝泉,酶定向进化的研究策略.中国医药工业杂志,2004.35(7)：p.436-441.
49. Wells, J.A., M. Vasser, and D.B. Powers, Cassette mutagenesis:an efficient method for generation of multiple mutations at defined sites. Gene,1985.34(2-3):p.315-23.
50. Russell, A.J. and A.R. Fersht, Rational modification of enzyme catalysis by engineering surface charge. Nature,1987.328(6130):p.496-500.
51. Schultz, S.C. and J.H. Richards, Site-saturation studies of beta-lactamase:production and characterization of mutant beta-lactamases with all possible amino acid substitutions at residue 71. Proc Natl Acad Sci U S A,1986.83(6):p.1588-92.
52. Kolkman, J.A. and W.P. Stemmer, Directed evolution of proteins by exon shuffling. Nat Biotechnol,2001.19(5):p.423-8.
53. Leung DW, C.E., Goeddel DV, A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique,1989.1(1):p.11-15.
54. Cadwell, R.C. and G.F. Joyce, Mutagenic PCR. PCR Methods Appl,1994.3(6):p. S136-40.
55. Kim, Y.W., et al., Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire. J Biol Chem,2004.279(41):p.42787-93.
56. Crameri, A., et al., DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature,1998.391(6664):p.288-91.
57. Kikuchi, M., K. Ohnishi, and S. Harayama, Novel family shuffling methods for the in vitro evolution of enzymes. Gene,1999.236(1):p.159-67.
58. Kikuchi, M., K. Ohnishi, and S. Harayama, An effective family shuffling method using single-stranded DNA. Gene,2000.243(1-2):p.133-7.
59. Zhao, H., et al., Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol,1998.16(3):p.258-61.
60. Ryu, K., et al., Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H2O2 stability. J Biotechnol,2008. 133(1):p.110-5.
61. Suen, W.C., et al., Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling. Protein Eng Des Sel,2004.17(2):p.133-40.
62. Bei, J., et al., Structure-based fragment shuffling of two fungal phytases for combination of desirable properties. J Biotechnol,2009.139(2):p.186-93.
63. Bessler, C., et al., Directed evolution of a bacterial alpha-amylase:toward enhanced pH-performance and higher specific activity. Protein Sci,2003.12(10):p.2141-9.
64. Kamondi, S., et al., Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling. Biochem Biophys Res Commun,2008.374(4):p.725-30.
65. Hao, J. and A. Berry, A. thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents. Protein Eng Des Sel,2004.17(9):p.689-97.
66. G, Z.Z.Y.Z.Z.L.L.Z., Cloning, DNA shuffling and expression of serine hydroxymethyhransferase gene from Eseherichia coli strain AB90054. Enzyme Microb Technol,2007.40(4):p.569-577.
67. Hsu, J.S., et al., Family shuffling of expandase genes to enhance substrate specificity for penicillin G. Appl Environ Microbiol,2004.70(10):p.6257-63.
68. Rosic, N.N., et al., Extending the diversity of cytochrome P450 enzymes by DNA family shuffling. Gene,2007.395(1-2):p.40-8.
69. Hu, H., et al., DNA shuffling of methionine adenosyltransferase gene leads to improved S-adenosyl-L-methionine production in Pichia pastor is. J Biotechnol,2009. 141(3-4):p.97-103.
70. Cesaro-Tadic, S., et al., Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library. Nat Biotechnol,2003.21(6):p.679-85.
71. Georgiou, G., et al., Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol,1997.15(1):p.29-34.
72. Mattheakis, L.C., R.R. Bhatt, and W.J. Dower, An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci U S A,1994. 91(19):p.9022-6.
73. Takahashi, T.T., R.J. Austin, and R.W. Roberts, mRNA display:ligand discovery, interaction analysis and beyond. Trends Biochem Sci,2003.28(3):p.159-65.
74. Xu, L., et al., Directed evolution of high-affinity antibody mimics using mRNA display. Chem Biol,2002.9(8):p.933-42.
2. Watkins, W.M., Biochemistry and Genetics of the ABO, Lewis, and P blood group systems. Adv Hum Genet,1980.10:p.1-136,379-85.
3. 章扬培,季守平,杨军,血型转变.中国实验血液学杂志,1998.6(2)：p.91-97.
4. Liu, Q.P., et al., Bacterial glycosidases for the production of universal red blood cells. Nat Biotechnol,2007.25(4):p.454-64.
5. Baeher, R.V. Biotechnology of blood. Biotechnology,1991.19:p.1-463.
6. Goldstein, J., et al., Group B erythrocytes enzymatically converted to group O survive normally in A, B, and O individuals. Science,1982.215(4529):p.168-70.
7. Kruskall, M.S., et al., Transfusion to blood group A and O patients of group B RBCs that have been enzymatically converted to group O. Transfusion,2000.40(11):p. 1290-8.
8. Zhu, A. and J. Goldstein, Cloning and functional expression of a cDNA encoding coffee bean alpha-galactosidase. Gene,1994.140(2):p.227-31.
9. Olsson, M.L. and H. Clausen, Modifying the red cell surface:towards an ABO-universal blood supply. Br J Haematol,2008.140(1):p.3-12.
10. Daniels, G and S.G. Withers, Towards universal red blood cells. Nat Biotechnol,2007. 25(4):p.427-8.
11. Lenny, L.L. and J. Goldstein, The production of group O cells. Biotechnology,1991. 19:p.75-100.
12. LaVecchio, J.A., A.D. Dunne, and A.S. Edge, Enzymatic removal of alpha-galactosyl epitopes from porcine endothelial cells diminishes the cytotoxic effect of natural antibodies. Transplantation,1995.60(8):p.841-7.
13. 杨军,宫锋,季守平,B→O血型转变工具酶α-半乳糖苷酶cDNA克隆及表达.中国生物化学与分子生物学报,2000.16(4)：p.438-442.
14. Stemmer, W.P., Rapid evolution of a protein in vitro by DNA shuffling. Nature,1994. 370(6488):p.389-91.
15. Harayama, S., Artificial evolution by DNA shuffling. Trends Biotechnol,1998.16(2):p. 76-82.
16. Chen, K. and F.H. Arnold, Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci U S A,1993.90(12):p.5618-22.
17. 曾家豫,杨国兵,廖世奇,生物酶的定向进化及其应用.卫生职业教育,2007.25(23)：p.155-159.
18. Kone, F.M., et al., Digital screening methodology for the directed evolution of transglycosidases. Protein Eng Des Sel,2009.22(1):p.37-44.
19. Ben-David, A., G. Shoham, and Y. Shoham, A universal screening assay for glycosynthases:directed evolution of glycosynthase XynB2(E335G) suggests a general path to enhance activity. Chem Biol,2008.15(6):p.546-51.
20. Landsteiner, K., Zur Kenntnis der antifermentativen, lytischen und agglutinierenden Wirkungen des Blutserums und der Lymphe. Zentralblatt Bakteriologie 1900(27):p. 357-362.
21. Donald, A.S., et al., The peptide moiety of human-blood-group active glycoproteins associated with the ABO and Lewis groups. Biochem J,1969.115(1):p.125-7.
22. Morgan, W.T. and W.M. Watkins, Genetic and biochemical aspects of human blood-group A-, B-, H-, Le-a- and Le-b-specificity. Br Med Bull,1969.25(1):p.30-4.
23. Morgan, W.T. and W.M. Watkins, Some aspects of the biochemistry of the human blood-group substances. Br Med Bull,1959.15(2):p.109-13.
24. Scott, M.D., et al., Chemical camouflage of antigenic determinants:stealth erythrocytes. Proc Natl Acad Sci U S A,1997.94(14):p.7566-71.
25. 张印则,红细胞抗原修饰的化学进展.中国输血杂志,2003.16(6)：p.435-437.
26. Abuchowski, A., et al., Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J Biol Chem,1977.252(11):p. 3578-81.
27. Fuertges F, A.A., The clinical efficacy of poly(ethyleneglycol) modified proteins. Control Release,1990(11):p.139-148.
28. Macchiarini, P., et al., Evidence of human non-alpha-galactosyl antibodies involved in the hyper acute rejection of pig lungs and their removal by pig organ perfusion. J Thorac Cardiovasc Surg,1998.116(5):p.831-43.
29. 季守平,刘泽澎,任会明,甲氧基聚乙二醇遮蔽红细胞表面抗原制备通用型血液的初步研究.中国输血杂志,2000.13(4)：p.223-227.
30. Hortin, G.L., H.T. Lok, and S.T. Huang, Progress toward preparation of universal donor red cells. Artif Cells Blood Substit Immobil Biotechnol,1997.25(5):p.487-91.
31. 檀英霞,王捷熙,章扬培,体外化学修饰红细胞——通用型血?.生命科学,2004.16(1).
32. 赵瑞东,王增祺,魏秀萍,红细胞ABO血型抗原表达及化学修饰.广州医药.38(4)：p.6-9.
33. 殷文劫,阮长耿,红细胞血型抗原修饰的研究与展望.苏州大学学报,2007.27(1)：p.68-72.
34. 季守平,刘泽澎,任会明,甲氧基聚乙二醇遮蔽红细胞表面抗原制备通用型血液的初步研究.中国输血杂志,2000.13(4)：p.223-227.
35. Lenny, L.L., et al., Multiple-unit and second transfusions of red cells enzymatically converted from group B to group O:report on the end of phase 1 trials. Transfusion, 1995.35(11):p.899-902.
36. Lenny, L.L., et al., Transfusions to group O subjects of 2 units of red cells enzymatically converted from group B to group O. Transfusion,1994.34(3):p. 209-14.
37. Zhu, A., et al., Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes. Arch Biochem Biophys,1996.327(2):p.324-9.
38.血型转换器.新技术：p.21.
39. 章扬培,杨军,高新,用基因重组α-半乳糖苷酶进行B→O血型改造.科学通报,2003.48(1)：p.43-47.
40. 高红伟,高新,鲍国强,基因重组α-半乳糖苷酶的制备工艺研究.生物技术通讯,2004.15(6)：p.566-569.
41. 鲍国强,高红伟,宫锋,基因重组α-半乳糖苷酶的生化性质研究.军事医学科学院院刊,2005.29(2)：p.139-141.
42. 宫峰,吕秋霜,由英,用α-半乳糖苷酶制备可供输注的人通用型O型红细胞.中国实验血液学杂志,2005.29(2)：p.139-141.
43. 刘泽澎,张成林,卢雁平,α-半乳糖苷酶酶解B型长臂猿红细胞回输A型长臂猿的研究.中国生物化学和分子生物学报,2004.20(2)：p.229-233.
44. 郁成雨,徐华,王立生,张建耕,章扬培,新型高比活力α-N-乙酰半乳糖胺酶应用于人红细胞.科学通报,2008.53(11)：p.1288-1295.
45. 徐华,郁成雨,章扬培,联合应用α-半乳糖苷酶和α-N-乙酰半乳糖胺酶实现红细胞AB→O血型转变.中国输血杂志,2008.21(12)：p.917-920.
46. 谢晚彬,谢和芳,蛋白质定向进化的研究技术及应用.中国生物工程杂志,2005：p.16-18.
47. 方柏山,郑媛媛,酶体外定向进化[I]——突变基因文库构建技术及其新进展.华侨大学学报,2004.25(4)：p.337-342.
48. 徐威,朱春宝,朱宝泉,酶定向进化的研究策略.中国医药工业杂志,2004.35(7)：p.436-441.
49. Wells, J.A., M. Vasser, and D.B. Powers, Cassette mutagenesis:an efficient method for generation of multiple mutations at defined sites. Gene,1985.34(2-3):p.315-23.
50. Russell, A.J. and A.R. Fersht, Rational modification of enzyme catalysis by engineering surface charge. Nature,1987.328(6130):p.496-500.
51. Schultz, S.C. and J.H. Richards, Site-saturation studies of beta-lactamase:production and characterization of mutant beta-lactamases with all possible amino acid substitutions at residue 71. Proc Natl Acad Sci U S A,1986.83(6):p.1588-92.
52. Kolkman, J.A. and W.P. Stemmer, Directed evolution of proteins by exon shuffling. Nat Biotechnol,2001.19(5):p.423-8.
53. Leung DW, C.E., Goeddel DV, A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique,1989.1(1):p.11-15.
54. Cadwell, R.C. and G.F. Joyce, Mutagenic PCR. PCR Methods Appl,1994.3(6):p. S136-40.
55. Kim, Y.W., et al., Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire. J Biol Chem,2004.279(41):p.42787-93.
56. Crameri, A., et al., DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature,1998.391(6664):p.288-91.
57. Kikuchi, M., K. Ohnishi, and S. Harayama, Novel family shuffling methods for the in vitro evolution of enzymes. Gene,1999.236(1):p.159-67.
58. Kikuchi, M., K. Ohnishi, and S. Harayama, An effective family shuffling method using single-stranded DNA. Gene,2000.243(1-2):p.133-7.
59. Zhao, H., et al., Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol,1998.16(3):p.258-61.
60. Ryu, K., et al., Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H2O2 stability. J Biotechnol,2008. 133(1):p.110-5.
61. Suen, W.C., et al., Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling. Protein Eng Des Sel,2004.17(2):p.133-40.
62. Bei, J., et al., Structure-based fragment shuffling of two fungal phytases for combination of desirable properties. J Biotechnol,2009.139(2):p.186-93.
63. Bessler, C., et al., Directed evolution of a bacterial alpha-amylase:toward enhanced pH-performance and higher specific activity. Protein Sci,2003.12(10):p.2141-9.
64. Kamondi, S., et al., Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling. Biochem Biophys Res Commun,2008.374(4):p.725-30.
65. Hao, J. and A. Berry, A. thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents. Protein Eng Des Sel,2004.17(9):p.689-97.
66. G, Z.Z.Y.Z.Z.L.L.Z., Cloning, DNA shuffling and expression of serine hydroxymethyhransferase gene from Eseherichia coli strain AB90054. Enzyme Microb Technol,2007.40(4):p.569-577.
67. Hsu, J.S., et al., Family shuffling of expandase genes to enhance substrate specificity for penicillin G. Appl Environ Microbiol,2004.70(10):p.6257-63.
68. Rosic, N.N., et al., Extending the diversity of cytochrome P450 enzymes by DNA family shuffling. Gene,2007.395(1-2):p.40-8.
69. Hu, H., et al., DNA shuffling of methionine adenosyltransferase gene leads to improved S-adenosyl-L-methionine production in Pichia pastor is. J Biotechnol,2009. 141(3-4):p.97-103.
70. Cesaro-Tadic, S., et al., Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library. Nat Biotechnol,2003.21(6):p.679-85.
71. Georgiou, G., et al., Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol,1997.15(1):p.29-34.
72. Mattheakis, L.C., R.R. Bhatt, and W.J. Dower, An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci U S A,1994. 91(19):p.9022-6.
73. Takahashi, T.T., R.J. Austin, and R.W. Roberts, mRNA display:ligand discovery, interaction analysis and beyond. Trends Biochem Sci,2003.28(3):p.159-65.
74. Xu, L., et al., Directed evolution of high-affinity antibody mimics using mRNA display. Chem Biol,2002.9(8):p.933-42.