人新基因EOLA1的原核表达、蛋白纯化及多克隆抗体制备
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摘要
脓毒症(sepsis)是多器官功能障碍(MODS)的前兆,是当前危重病医学面临的难题之一,它也是导致严重烧伤、创伤患者死亡的主要原因之一;其发病机制与内毒素(LPS)激活血管内皮细胞诱发并加重全身炎症反应密切相关。脓毒症的病理生理机制复杂,现行的治疗措施有限,因此我们深入研究其发病机制就显得尤为重要。
近年来,由于血管内皮细胞是内毒素作用的直接靶细胞之一,国内外对LPS激活内皮细胞的作用途径、特异受体、胞内信号转导等方面研究较多。本研究室前期应用抑制消减杂交(SSH)和cDNA末端扩增技术(RACE),从LPS刺激后的人脐静脉内皮细胞中克隆到一个人类新基因全长cDNA序列,经Northern blot验证后作为人类新基因被GenBank全长序列库所接受(No.AY074889)。因其在受LPS刺激的内皮细胞中上调表达,故暂时命名为内皮高表达脂多糖相关因子1(endothelial-overexpressed lipopolysaccharide-associated factor 1,EOLA1)。
目前,国外尚无关于该基因的相关研究,本实验室对EOLA1基因做了一些前期研究工作,并经生物信息学分析显示:该基因全长1404个bp,其定位于染色体Xq27.4,编码蛋白由158个氨基酸组成,分子量17.89kDa,等电点6.43,亲水性好,为可溶性蛋白。其二级结构含α螺旋、β片层结构和β转角,并形成一个螺旋—转角—螺旋(HTH)基序。EOLA1氨基酸序列包含N-糖基化位点、PKC磷酸化位点和酪氨酸激酶2磷酸化位点。
多组织Northern blot显示EOLA1基因除在LPS刺激的内皮细胞上调表达外,在骨胳肌、心脏、肾脏、肝脏和胎盘有较强的表达,在结肠、小肠、脾脏有较弱的表达,而在脑、胸腺、肺和外周白细胞则未见表达。在7种癌细胞株上,显示在早幼粒白血病系HL-60、海拉细胞系S3、慢性骨髓性白血病系K-562和肺癌细胞系A549都有表达,而在成淋巴细胞瘤性白血病MOLT-4和结直肠腺癌SW480表达水平最高,但在黑素瘤G-361无表达。在经过G418压力筛选后的稳定表达EGFP-EOLA1(enhanced green fluorescent protein-EOLA1)融合蛋白的ECV304细胞株中发现EOLA1蛋白为全细胞分布。酵母双杂交显示EOLA1蛋白与细胞内金属硫蛋白相互作用,可能参与细
Sepsis is an auspice of multiple organ dysfunctional syndrome (MODS) and an intractable problem, which is facing with in critical and serious illness medicine. Sepsis is also the one of main causes of death in serious burn and traumatic patients. EC is one kind of the main target cells that are directly activated by LPS, and effect on EC by LPS with regard to signal transmitting and special receptor and route of action has been studied more. The EOLA1 gene (a human novel gene, GenBank Accession No. AY074889) was cloned in our laboratory by the effective technique of mRNA differential display (comparing the differential gene expression between normal ECV304 and treated by LPS). Using the SMART RACE technique, the full-length cDNA of the EOLA1 gene was cloned. It contains 1404 nucleotides, 474 nucleotides of the open reading frame that predicts 158 amino acids. The genomic DNA contains 5 exons, spans about 6294 bps, and is mapped to human chromosome Xq27.4. Analyzing by bioinformatics methods found that EOLA1 secondary structure contains α -helix, β -Lamellosa and β -turn, and a helix-turn-helix (HTH) motif. This information indicated that EOLAl may be as a transcription factor, which play an important role in process of activating Human EC.Northern blot with some kinds of Human tissues and carcinoma cells revealed an extended expression profile of EOLA1. It was highly expressed in skeletal muscle, liver, placenta and lowly expressed in the spleen, heart and no expressed in brain, colon, small intestine, thymus, lung and peripheral blood leukocyte; The yeast two-hybrid experiment has showed the interaction of EOLA1 and MT2A which may play some roles of apoptosis and growth and anti-inflammation in human EC. Now the biological function of EOAL1 is unknown, so preparing anti-EOLA1 antibody is very important to functional studying of EOLA1 in the future.The open reading frame of EOLA1 gene was cloned by RT-PCR from human ECV304 cells, and was judged as 500bp length by agarose gel electrophoresis. Recombinant
plasmid was successfully constructed and identified by colony PCR and sequence measurement. Recombinant EOLAl protein was successfully expressed in BL21(DE3)plysS and the expressed protein in E.coli mainly exist in inclusion body form. The molecular weight of EOLAl is about 20x103 by SDS-PAGE analysing and Western blot. The optimization of induction conditions which include Incubate at 37°C for 4h, E.coli BL21(DE3)plysS, lmmol/L IPTG and 100 u g/ml ampicillin was founded through experiment. The target protein has been expressed in large-scale under above induction conditions: 9.6g of the cells collected by centrifugation from induced 2000ml culture and 0.76g of inclusion bodies were collected after primary purification including destruction of bacteria, washing-up and isolation. 100ml of target protein solution with 124.16ug/ml concentration was obtained after immobilized metal affinity chromatography and renaturation. Peptide mass fingerprinting show the matched peptides cover 32% (56/172 AA's) of the theoretic EOLAl protein. The anti-EOLAl polyclonal antibody was collected through BALB/c rat immunization and identified by Western-blot. ELISA showed its efficiency has arrived at 1:10000.In conclusion, the opening read frame of EOLAl has been cloned by RT-PCR from ECV304 cells and identified by sequence analysis, which further confirms the veracity and facticity of EOLAl. EOLAl recombinant protein can be expressed in E.coli largely in inclusion body form. Anti-EOLAl polyclonal antibody that is produced by our experiment can detect expression of nature EOLAl in ECV304 cells.
近年来,由于血管内皮细胞是内毒素作用的直接靶细胞之一,国内外对LPS激活内皮细胞的作用途径、特异受体、胞内信号转导等方面研究较多。本研究室前期应用抑制消减杂交(SSH)和cDNA末端扩增技术(RACE),从LPS刺激后的人脐静脉内皮细胞中克隆到一个人类新基因全长cDNA序列,经Northern blot验证后作为人类新基因被GenBank全长序列库所接受(No.AY074889)。因其在受LPS刺激的内皮细胞中上调表达,故暂时命名为内皮高表达脂多糖相关因子1(endothelial-overexpressed lipopolysaccharide-associated factor 1,EOLA1)。
目前,国外尚无关于该基因的相关研究,本实验室对EOLA1基因做了一些前期研究工作,并经生物信息学分析显示:该基因全长1404个bp,其定位于染色体Xq27.4,编码蛋白由158个氨基酸组成,分子量17.89kDa,等电点6.43,亲水性好,为可溶性蛋白。其二级结构含α螺旋、β片层结构和β转角,并形成一个螺旋—转角—螺旋(HTH)基序。EOLA1氨基酸序列包含N-糖基化位点、PKC磷酸化位点和酪氨酸激酶2磷酸化位点。
多组织Northern blot显示EOLA1基因除在LPS刺激的内皮细胞上调表达外,在骨胳肌、心脏、肾脏、肝脏和胎盘有较强的表达,在结肠、小肠、脾脏有较弱的表达,而在脑、胸腺、肺和外周白细胞则未见表达。在7种癌细胞株上,显示在早幼粒白血病系HL-60、海拉细胞系S3、慢性骨髓性白血病系K-562和肺癌细胞系A549都有表达,而在成淋巴细胞瘤性白血病MOLT-4和结直肠腺癌SW480表达水平最高,但在黑素瘤G-361无表达。在经过G418压力筛选后的稳定表达EGFP-EOLA1(enhanced green fluorescent protein-EOLA1)融合蛋白的ECV304细胞株中发现EOLA1蛋白为全细胞分布。酵母双杂交显示EOLA1蛋白与细胞内金属硫蛋白相互作用,可能参与细
Sepsis is an auspice of multiple organ dysfunctional syndrome (MODS) and an intractable problem, which is facing with in critical and serious illness medicine. Sepsis is also the one of main causes of death in serious burn and traumatic patients. EC is one kind of the main target cells that are directly activated by LPS, and effect on EC by LPS with regard to signal transmitting and special receptor and route of action has been studied more. The EOLA1 gene (a human novel gene, GenBank Accession No. AY074889) was cloned in our laboratory by the effective technique of mRNA differential display (comparing the differential gene expression between normal ECV304 and treated by LPS). Using the SMART RACE technique, the full-length cDNA of the EOLA1 gene was cloned. It contains 1404 nucleotides, 474 nucleotides of the open reading frame that predicts 158 amino acids. The genomic DNA contains 5 exons, spans about 6294 bps, and is mapped to human chromosome Xq27.4. Analyzing by bioinformatics methods found that EOLA1 secondary structure contains α -helix, β -Lamellosa and β -turn, and a helix-turn-helix (HTH) motif. This information indicated that EOLAl may be as a transcription factor, which play an important role in process of activating Human EC.Northern blot with some kinds of Human tissues and carcinoma cells revealed an extended expression profile of EOLA1. It was highly expressed in skeletal muscle, liver, placenta and lowly expressed in the spleen, heart and no expressed in brain, colon, small intestine, thymus, lung and peripheral blood leukocyte; The yeast two-hybrid experiment has showed the interaction of EOLA1 and MT2A which may play some roles of apoptosis and growth and anti-inflammation in human EC. Now the biological function of EOAL1 is unknown, so preparing anti-EOLA1 antibody is very important to functional studying of EOLA1 in the future.The open reading frame of EOLA1 gene was cloned by RT-PCR from human ECV304 cells, and was judged as 500bp length by agarose gel electrophoresis. Recombinant
plasmid was successfully constructed and identified by colony PCR and sequence measurement. Recombinant EOLAl protein was successfully expressed in BL21(DE3)plysS and the expressed protein in E.coli mainly exist in inclusion body form. The molecular weight of EOLAl is about 20x103 by SDS-PAGE analysing and Western blot. The optimization of induction conditions which include Incubate at 37°C for 4h, E.coli BL21(DE3)plysS, lmmol/L IPTG and 100 u g/ml ampicillin was founded through experiment. The target protein has been expressed in large-scale under above induction conditions: 9.6g of the cells collected by centrifugation from induced 2000ml culture and 0.76g of inclusion bodies were collected after primary purification including destruction of bacteria, washing-up and isolation. 100ml of target protein solution with 124.16ug/ml concentration was obtained after immobilized metal affinity chromatography and renaturation. Peptide mass fingerprinting show the matched peptides cover 32% (56/172 AA's) of the theoretic EOLAl protein. The anti-EOLAl polyclonal antibody was collected through BALB/c rat immunization and identified by Western-blot. ELISA showed its efficiency has arrived at 1:10000.In conclusion, the opening read frame of EOLAl has been cloned by RT-PCR from ECV304 cells and identified by sequence analysis, which further confirms the veracity and facticity of EOLAl. EOLAl recombinant protein can be expressed in E.coli largely in inclusion body form. Anti-EOLAl polyclonal antibody that is produced by our experiment can detect expression of nature EOLAl in ECV304 cells.
引文
1. Collins FS, Green ED, Guttmacher AE, et al. A version for the future of genomics research. Nature, 2003, 402: 8 35-47
2. Wilkins, MR, Sanchez JC, Gooley AA, et al. Progress with proteome projects: why all proteins expressed by a genome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1995, 13: 19-50
3. Fields S. Proteomics in genome land. Science, 2001, 291(5507): 1221-1224
4. Pandey A, Mann M. Proteomics to study genomes. Nature, 2000, 405 (6788): 837-846
5.盛志勇,姚咏明。脓毒症研究的现状与展望。解放军医学杂志,1999,24:79—82
6. Avery BN, John CM. Sepsis, SIRS and MODS: what is in a name? World J Surg, 1996, 20: 386
7. Skurnik-M, Bengoechea-J-A. The biosynthesis and biological role of lipopolysaccharide O-antigens of pathogenic Yersiniae. Carbohydr-Res. 2003 Nov 14; 338(23): 2521-2529
8. Landmann R, Muller B, Aimmerli W. CD14, new aspects of ligandand signal diversity. Microbes Infect, 2000, 2: 295-304
9. Lien E, Sellati TJ, Yoshimura A, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem, 1999, 274: 33419-33425
10. Shimazu R, Akashi S, Ogata H, et al. MD-2, A molecule that confers lipopolysaccharide responsiveness on toll-like receptor 4. J Exp Med, 1999, 189: 1777-1782
11. Ulevitch RJ, Tobias PS. Recognition of endotoxin bycells leading to transmembrane signalling. Curr Opinimmunol, 1994, 6: 125-130
12.梁自文,杨宗城,罗向东。人类新基因eolal全长序列的克隆。第三军医大学学报,2002,24(3):2
13.梁自文,罗向东,杨宗城。应用抑制消减杂交克隆内皮细胞内毒素刺激后相关基因。解放军医学杂志,2002,27(4):336—338
14.梁自文,杨宗城,罗向东。人类内皮化相关新基因EOLA1的发现及初步研究。解 放军医学杂志,2003,28(5):422—424
15. Ziwen Liang, Zongcheng Yang. Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation Biochemical and Biophysical Research Communications 325 (2004) 798-802
16. Peltonen L, Mekusick VA. Dissecting human disease in the postgenomic era. Science, 2001, 291: 1224-1229
17.陈竺,强伯勤,方福德主编。基因组科学与人类疾病。北京:科学出版社,2001
18. Studier F W, Rosenberg A H, Dunn J J et, al. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990, 185: 60-89
19.司徒镇强,吴军正,主编。细胞培养。北京,世界图书出版公司,1996:34-39
20.颜子颖,王海林 译。精编分子生物学实验指南。第一版,北京:科学出版社,1998
21.黎鳌,杨宗城。黎鳌烧伤学。上海:上海科学技术出版社,2001.11:67—69
22. Hunt B J, Jurd KM. Endothelial cell activation: a central pathophysiol-ogical protess. BMJ, 1998, 316: 1328
23. Pugin J, Ulevitch RJ, Tobias PS. Activation of endothelial cells by endotoxin: direct versus indirect pathways and the role of CD14. Prog Clin Biol Res, 1995, 392: 369
24.罗向东,石富胜,王晓军,等。LPS对血管内皮细胞损伤的直接损伤作用的初步研究。第三军医大学学报,2003,18(25):1609—1612
25. Chan EL, Haudek SB, Giroir BP et al. Human coronary endothelial cell activation by endotoxin is characterized by NF-kappaB activation and TNF-alpha synthesis. Shock, 2001, 16(5): 349
26.蒋建新,朱佩芳,王正国。内毒素的跨膜信号转导及在全身炎症反应综合征中的作用。解放军医学杂志,2002,1(27):1—4
27. Treisman—J, Harris—E, Wilson—D et al. The homeodomain: a new face for the helix-turn-helix? Bioessays. 1992 Mar; 14(3): 145-10
28. K Yura, S Tomoda and M Go. Repeat of a helix-turn-helix module in DNA-binding proteins. Protein Eng. 1993 Aug; 6(6): 621-628
29. Haun,-R-S; Serventi,-I-M; Moss,-J. Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. Biotechniques. 1992 Oct; 13(4): 515-518
30. Zhang,-X, Studier,-F-W. Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J-Mol-Biol. 1997 May 30; 269(1): 10-27
31. Lyakhov,-D-L, He,-B, Zhang,-X. Pausing and termination by bacteriophage T7 RNA polymerase. J-Mol-Biol. 1998 Jul 10; 280(2): 201-13
32. Zhang, -X, Studier, -F-W. Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection. J-Mol-Biol. 2004 Jul 16; 340 (4): 707-30
33. Preibisch, -G; Ishihara, -H; Tripier, -D; et al Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coll. Gene. 1988 Dec 10; 72(1-2): 179-86
34. Smith S, Hailing S M, Expression of canine parvovirus-beta-galactosidase fusion proteins in Escherichia coli. Gene. 1984 Sep; 29(3): 263-9
35. Kim J S, Raines R T. Peptide tags for a dual affinity fusion system. Anal-Biochem. 1994 May 15; 219(1): 165-6
36. Wemer M H, Clore G M, Refolding proteins by gel filtration chromatography. FEBS Lett, 1994, 345(2)
37. Humphery-Smith I, Cordwell S J, Blackstock W P. Proteome research: Complementarity and limitations with respect to the RNA and DNA worlds. Electrophoresis, 1997, 18(8): 1217-1242
38. Wilkins M R, Sanchez J C, Gooley A A, Appel R D, HumpherySmith I, Hochstrasser D F, Willioms K L. Progress with proteome projects: Why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1996, 13: 19-50
39. Wilkins M R, Gooley A A. In: Wilkins M R, Williams KL, Appel R D, Hochstrasser D F eds. Proteome Research: New frontiers in functional genomics, Berlin: Springer-Verlag, 1997, 35-61
40. Wise M J, Littlejohn T G, Humphery-Smith I. Peptide mass fingerprinting and the ideal covering set for protein characterization. Electrophoresis, 1997, 18(8): 1399-1409
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42. Anton P. J. Middelberg. Preparative protein refolding. Trend in Biotechnology, 2002, 20(10): 437-443
43. G. Kohler and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256 (1975), pp. 495-497
44.金伯泉。细胞和分子免疫学。北京:科学出版社,2001:439—443
45. Stephen A. Wring, et al, Shorter development of immunoassay for drugs: application of the novel RIMMS technique enables rapid production of monoclonal antibodies to ranitidine. Journal of Pharmaceutical and Biomedical Analysis 19 (1999) 695-707
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2. Wilkins, MR, Sanchez JC, Gooley AA, et al. Progress with proteome projects: why all proteins expressed by a genome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1995, 13: 19-50
3. Fields S. Proteomics in genome land. Science, 2001, 291(5507): 1221-1224
4. Pandey A, Mann M. Proteomics to study genomes. Nature, 2000, 405 (6788): 837-846
5.盛志勇,姚咏明。脓毒症研究的现状与展望。解放军医学杂志,1999,24:79—82
6. Avery BN, John CM. Sepsis, SIRS and MODS: what is in a name? World J Surg, 1996, 20: 386
7. Skurnik-M, Bengoechea-J-A. The biosynthesis and biological role of lipopolysaccharide O-antigens of pathogenic Yersiniae. Carbohydr-Res. 2003 Nov 14; 338(23): 2521-2529
8. Landmann R, Muller B, Aimmerli W. CD14, new aspects of ligandand signal diversity. Microbes Infect, 2000, 2: 295-304
9. Lien E, Sellati TJ, Yoshimura A, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem, 1999, 274: 33419-33425
10. Shimazu R, Akashi S, Ogata H, et al. MD-2, A molecule that confers lipopolysaccharide responsiveness on toll-like receptor 4. J Exp Med, 1999, 189: 1777-1782
11. Ulevitch RJ, Tobias PS. Recognition of endotoxin bycells leading to transmembrane signalling. Curr Opinimmunol, 1994, 6: 125-130
12.梁自文,杨宗城,罗向东。人类新基因eolal全长序列的克隆。第三军医大学学报,2002,24(3):2
13.梁自文,罗向东,杨宗城。应用抑制消减杂交克隆内皮细胞内毒素刺激后相关基因。解放军医学杂志,2002,27(4):336—338
14.梁自文,杨宗城,罗向东。人类内皮化相关新基因EOLA1的发现及初步研究。解 放军医学杂志,2003,28(5):422—424
15. Ziwen Liang, Zongcheng Yang. Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation Biochemical and Biophysical Research Communications 325 (2004) 798-802
16. Peltonen L, Mekusick VA. Dissecting human disease in the postgenomic era. Science, 2001, 291: 1224-1229
17.陈竺,强伯勤,方福德主编。基因组科学与人类疾病。北京:科学出版社,2001
18. Studier F W, Rosenberg A H, Dunn J J et, al. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990, 185: 60-89
19.司徒镇强,吴军正,主编。细胞培养。北京,世界图书出版公司,1996:34-39
20.颜子颖,王海林 译。精编分子生物学实验指南。第一版,北京:科学出版社,1998
21.黎鳌,杨宗城。黎鳌烧伤学。上海:上海科学技术出版社,2001.11:67—69
22. Hunt B J, Jurd KM. Endothelial cell activation: a central pathophysiol-ogical protess. BMJ, 1998, 316: 1328
23. Pugin J, Ulevitch RJ, Tobias PS. Activation of endothelial cells by endotoxin: direct versus indirect pathways and the role of CD14. Prog Clin Biol Res, 1995, 392: 369
24.罗向东,石富胜,王晓军,等。LPS对血管内皮细胞损伤的直接损伤作用的初步研究。第三军医大学学报,2003,18(25):1609—1612
25. Chan EL, Haudek SB, Giroir BP et al. Human coronary endothelial cell activation by endotoxin is characterized by NF-kappaB activation and TNF-alpha synthesis. Shock, 2001, 16(5): 349
26.蒋建新,朱佩芳,王正国。内毒素的跨膜信号转导及在全身炎症反应综合征中的作用。解放军医学杂志,2002,1(27):1—4
27. Treisman—J, Harris—E, Wilson—D et al. The homeodomain: a new face for the helix-turn-helix? Bioessays. 1992 Mar; 14(3): 145-10
28. K Yura, S Tomoda and M Go. Repeat of a helix-turn-helix module in DNA-binding proteins. Protein Eng. 1993 Aug; 6(6): 621-628
29. Haun,-R-S; Serventi,-I-M; Moss,-J. Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. Biotechniques. 1992 Oct; 13(4): 515-518
30. Zhang,-X, Studier,-F-W. Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J-Mol-Biol. 1997 May 30; 269(1): 10-27
31. Lyakhov,-D-L, He,-B, Zhang,-X. Pausing and termination by bacteriophage T7 RNA polymerase. J-Mol-Biol. 1998 Jul 10; 280(2): 201-13
32. Zhang, -X, Studier, -F-W. Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection. J-Mol-Biol. 2004 Jul 16; 340 (4): 707-30
33. Preibisch, -G; Ishihara, -H; Tripier, -D; et al Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coll. Gene. 1988 Dec 10; 72(1-2): 179-86
34. Smith S, Hailing S M, Expression of canine parvovirus-beta-galactosidase fusion proteins in Escherichia coli. Gene. 1984 Sep; 29(3): 263-9
35. Kim J S, Raines R T. Peptide tags for a dual affinity fusion system. Anal-Biochem. 1994 May 15; 219(1): 165-6
36. Wemer M H, Clore G M, Refolding proteins by gel filtration chromatography. FEBS Lett, 1994, 345(2)
37. Humphery-Smith I, Cordwell S J, Blackstock W P. Proteome research: Complementarity and limitations with respect to the RNA and DNA worlds. Electrophoresis, 1997, 18(8): 1217-1242
38. Wilkins M R, Sanchez J C, Gooley A A, Appel R D, HumpherySmith I, Hochstrasser D F, Willioms K L. Progress with proteome projects: Why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1996, 13: 19-50
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