草鱼生长激素基因的克隆及原核表达研究
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摘要
本研究选用草鱼(Ctenspharynogodom idellus)作为实验材料，从草鱼脑垂体中提取总RNA。设计合成了一对草鱼生长激素基因的特异性引物，上游引物加入BamHI酶切位点；下游引物加入EcoRI酶切位点。应用逆转录-聚合酶链式反应(RT-PCR)技术克隆得到编码草鱼生长激素(CGH)的基因cDNA，并定向克隆到pUC18载体上。正反向测序结果分析表明：克隆的草鱼生长激素基因cDNA全长669bp，开放阅读框(ORF)包含633个碱基，编码210个氨基酸。分子量为23.6KD，等电点为6.28；疏水氨基酸占43.3％，亲水氨基酸占32.4％，碱性氨基酸占12.4％，酸性氨基酸占11.9％。将得到的序列在GenBank和EMBL数据库中进行了同源比较，结果显示：本研究克隆到的草鱼GH基因与GenBank中登记的X60474草鱼GH基因有12个碱基的差异，编码的氨基酸有3个氨基酸残基的差异，同源性为98％，影响蛋白质高级结构的保守二硫键为2个。证明本实验成功克隆到了草鱼生长激素基因的编码序列。
采用DNA重组技术将已克隆到的草鱼生长激素(CGH)cDNA片段定向插入融合表达载体pGEX-4T-1质粒中，转化大肠杆菌BL21，用菌落PCR和质粒PCR方法筛选阳性克隆；以BamHⅠ和EcoRⅠ酶切鉴定重组质粒。以IPTG诱导融合蛋白表达，并作RT-PCR及SDS-PAGE分析。RT-PCR分析确证重组质粒在大肠杆菌中能正确转录；SDS-PAGE分析证明诱导重组质粒表达了融合蛋白，分子量约为50KD，与预期的26 KD的GST带和23.6KD的草鱼生长激素基因编码蛋白质构成的融合蛋白大小一致。融合蛋白表达量占总蛋白量的20％。实验证明得到高效表达草鱼GH的重组质粒，命名为pGCGH。
本研究克隆到草鱼生长激素基因cDNA序列，并首次选用融合表达载体作为鱼类生长激素基因的原核表达工具，成功构建了重组CGH原核表达质粒，实现了CGH的高效表达。为进一步开展草鱼生长激素的应用和生物活
四川大学硕士学位论文草鱼生长激素墓因的克隆及原核表达研究
性检测分析、真核表达研究及研制新型调节鱼类生长的基因制剂的应用奠定
了坚实的基础。
Total RNA was isolated from grass carp (Ctenspharynogodom idellus) pituitary. The cDNA encoding growth hormone (GH) peptide was amplified by reverse transcription polymerase chain reaction (RT-PCR) method using isolated total RNA as template. The amplified cDNA fragment was inserted to plasmid pUC 18. The sequencing result shows that length of CGH cDN A fragment is 669bp. ORF include of 633bp, encoding 210 amino acids with molecular 23.6 KDa weight and pI is 6.28. CGH peptide include 12.4% Basic Amino Acids, 11.9% Acidic Amino Acids, 32.4% Hydrophilic Amino Acids, 43.3% Hydrophonic Amino Acids. By blasting the homologous sequences in GenBank databases, the sequence of grass carp GH cDNA from pituitary is 98% homologous compared with the previously cloned GH cDNA of grass carp .
The CGH cDNA fragment was inserted into pGEX-4T-l to construct the expression plasmid. The recombinant plasmid was digested by BamH I and EcoR I
to identify whether the CGH cDNA fragment was inserted into the plasmid, the pGCGH was transformed into E.coli BL21 competent cells. The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE. The results were found that the specific 670bp DNA bases of CGH was detected by RT-PCR in the recombinant bacteria and a new protein band was found in SDS-PAGE with molecular mass of about 50 KDa which is consisted of a 23.6 KDa protein deduced from the CGH gene sequence and GST (26 KDa). These indicated that the grass carp GH gene was correctly transcribed and translated in the recombinant bacteria, and could be employed to further develop a novel growth-regulating reagents to promote the health development offish.
采用DNA重组技术将已克隆到的草鱼生长激素(CGH)cDNA片段定向插入融合表达载体pGEX-4T-1质粒中，转化大肠杆菌BL21，用菌落PCR和质粒PCR方法筛选阳性克隆；以BamHⅠ和EcoRⅠ酶切鉴定重组质粒。以IPTG诱导融合蛋白表达，并作RT-PCR及SDS-PAGE分析。RT-PCR分析确证重组质粒在大肠杆菌中能正确转录；SDS-PAGE分析证明诱导重组质粒表达了融合蛋白，分子量约为50KD，与预期的26 KD的GST带和23.6KD的草鱼生长激素基因编码蛋白质构成的融合蛋白大小一致。融合蛋白表达量占总蛋白量的20％。实验证明得到高效表达草鱼GH的重组质粒，命名为pGCGH。
本研究克隆到草鱼生长激素基因cDNA序列，并首次选用融合表达载体作为鱼类生长激素基因的原核表达工具，成功构建了重组CGH原核表达质粒，实现了CGH的高效表达。为进一步开展草鱼生长激素的应用和生物活
四川大学硕士学位论文草鱼生长激素墓因的克隆及原核表达研究
性检测分析、真核表达研究及研制新型调节鱼类生长的基因制剂的应用奠定
了坚实的基础。
Total RNA was isolated from grass carp (Ctenspharynogodom idellus) pituitary. The cDNA encoding growth hormone (GH) peptide was amplified by reverse transcription polymerase chain reaction (RT-PCR) method using isolated total RNA as template. The amplified cDNA fragment was inserted to plasmid pUC 18. The sequencing result shows that length of CGH cDN A fragment is 669bp. ORF include of 633bp, encoding 210 amino acids with molecular 23.6 KDa weight and pI is 6.28. CGH peptide include 12.4% Basic Amino Acids, 11.9% Acidic Amino Acids, 32.4% Hydrophilic Amino Acids, 43.3% Hydrophonic Amino Acids. By blasting the homologous sequences in GenBank databases, the sequence of grass carp GH cDNA from pituitary is 98% homologous compared with the previously cloned GH cDNA of grass carp .
The CGH cDNA fragment was inserted into pGEX-4T-l to construct the expression plasmid. The recombinant plasmid was digested by BamH I and EcoR I
to identify whether the CGH cDNA fragment was inserted into the plasmid, the pGCGH was transformed into E.coli BL21 competent cells. The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE. The results were found that the specific 670bp DNA bases of CGH was detected by RT-PCR in the recombinant bacteria and a new protein band was found in SDS-PAGE with molecular mass of about 50 KDa which is consisted of a 23.6 KDa protein deduced from the CGH gene sequence and GST (26 KDa). These indicated that the grass carp GH gene was correctly transcribed and translated in the recombinant bacteria, and could be employed to further develop a novel growth-regulating reagents to promote the health development offish.
引文
1. Agellon L B, Emery C J, Jones J M. Promotion of rapid growth of rainbow trout by a recombinant fish growth hormone [J]. Can J Fish Aquat Sci, 1988, 45: 146～151.
2. Ber R, Daniel V. Structure and sequence of the growth hormone-encoding gene from Tilapia nilolica. Gene, 1992, 113: 245.
3. Chen S L, Deng W T, Liu X T, et al. Isolation and purification of growth hormone from grass carp(Ctenspharynogodom idellus) and preparation of antibody against the gcGH [J]. Acta Zoologica Sinica, 1995, 41: 282～290.
4. Chen S L, Hong Y H, Schartl M. Lack of ultraviolet light inducibility of the medakafish (Oryzias latipes) tumor suppress gene p53 [J]. Gene, 2001, 264: 197～203.
5. Chen S L, Yang F, He L, et al. Generation and characterization of monoclonal antibodies against grass carp (Ctenspharynogodom idellus) growth hormone [J]. Asian Fisheries Science, 1996, 9: 183～189.
6. Chen S L. Progress and prospect of gene engineering of fish peptide hormone [J]. J Fish China, 1993, 17: 264～270.
7. Clark W C, Farmer S W, Hartwell K M. Gene. Comp. Endocrinol., 1997, 27: 174.
8. Degani G, Gallagher M L. J. Fish. Aquat. Sci., 1985, 42: 185.
9. Donaldson E M, Fagerlund U H M, Higgs D A, McBride J R. Hormonal enhancement of growth. Fish Physiol, NewYork: Academic Press, 1979, 8: 455～597.
10. Duan C, Hirano T. An guillajaponica. Aquaculture, 1991, 95: 179.
11. Fine M, Sakal E, Vashdi D, et al. Recombinant carp (Cyprinus carp) growth hormone: expression, purification and determination of biological activity in vitro and in vivo [J]. Gene Comp Endocrinol, 1993, 89: 51～61.
12. Fu C, Cui Y, Hung S S O, Zhu Z. J. Fish. Bio., 1998, 53: 115.
13. Funkenstein B, et al. Cloning and sequencing of the Glithead seabream (Sparus aurata) growth homone-encodong cDNA. Gene, 1991, 103: 343.
14. Georgopoulou U, Sire M F, Vernier J M. Immunological demon-stration of intestinal absorption and digestion of protein macro-molecules in the trout (Salmogairdneri). Cell Tissue. Res, 1986, 245: 387～395.
15. Gill J A, Sumter J P, Donaldson E M, Dye H M, Souza L M, Berg T, Wypych J, Langley K. Recombinant chicken and bovine growth hormones accelerate growth in aquacultured juvenile Pacific salmon Oncorhynchus kisutch. Biotechnology, 1985, 3: 643～646.
16. Hertz Y et al. Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus carpio) and its enhancement by deoxycholate. J. Comp. Physiol., 1991, 161B: 159～163.
17. Hew C L, Trinh K Y, Du S J, et al. Molecular cloning and expression of salmon pituitary hormone [J]. Fish Physiol Biochem, 1989, 7: 375～380.
18. Ho W K K, et al. Cloning of the grass carp growth hormone cDNA. Biochem. Biophys. Res. Commun., 1990, 161: 1239.
19. Hong Y, Schartl M. Seqence of the growth hormone gene from the silver carp and evolution of GH genes in vertebrates [J]. Biochem Biophys Acta, 1993, 1174: 285～288.
20. Koren Y, Sarid S, Ber R, Daniel V. Carp growth hormone; molecular cloning and sequencing of cDNA. Gene, 1989,77: 309～315.
21. Lebail P Y, Sire M F, Vernier J M. Intestinal transfer for growth hormone into the circulatory system of the rainbow trout Salmo gairdneri: Interference by granule cell. J. Exp. Zool, 1989, 251: 101～107.
22. Marchant T A, Peter R E, Seasonal variations in body growth rates and circulation levels of growth hormones in the gold fish, Carassius auratus [J]. J Exp. Zool, 1986, 237～239.
23. Mclean E, Donaldson E M, Dye H M, Souza L M. Growth acceleration of coho salmon (Oncorhynchus kisutch) following oral administration of recombinant bovine somatotropin. Aquaculture, 1990, 91: 197～203.
24. Mclean E, Donaldson E M. The role of growth hormone in growth of poikilotherms. In: Schriebman M P, Scanes C G, Pang P K T, eds. The Endocrinology of Growth, Development and Metabolism in Vertebrates. SanDiego: Academic Press, 1993, 43～71.
25. Miler W L, Eberhardt N L. Structure and evolution of growth hormone gene family. Endocrine Rev., 1983, 4: 97.
26. Moore D D, et al. Structure, expression and evoluticn of growth hormone genes. Rec. Proc. Horm. Res, 1982, 38: 197.
27. Moriyama S, Yamamoto H, Sugimoto S et al. Oral administration of recombinant salmon
growth hormone to rainbow trout, Oneorhynehus mykiss. Aquaeulture, 1993, 112: 99～106.
28. Rentier-Delrue F, Swennen D et al. Tilapia growth hormone: Molecular cloning of cDNA and expression in Escherichia coli. DNA, 1989, 8: 271～278.
29. Saito A, et al. Molecular cloning of eel growth hormone cDNA and its expression in Escherichia coli. Gene, 1988, 73:545.
30. Sambrook J, et al. Molecular cloning: Alaboratory Manual. 2nded, NewYork: Cold Spring Harbor Laboratory Press, 1989.
31. Sato N, et al. Molecular cloning and nucleotide sequence of tuna growth hormone Cdna. Biochem. Biophys. Acta., 1988, 949: 35.
32. Schoepfer R. The pRSET fa mily of T7 promoter expression vectors for Escherichia coli[J]. Gene, 1993, 124: 83～85.
33. Sekine S, et al., Isolation and characterization of a novel growth hormone cDNA from salmon (Oncohynchus keta). Biochem. Biophys. Acta., 1989, 1009: 117.
34. Sekine S, Mizukami T, Nishi T et al. Cloning and expressing of cDNA for salmon growth hormone in Escherichia coli. Proc. Natl. Acad. Sci. USA, 1985, 82: 4306～4310.
35. Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15; 67(1): 31-40.
36. Tsai H J, Chen H M, Lo C F. Sceretory synthesis of active recombinant fish growth hormone by insect cells using a Bsculovirus Vector. Can. J. Fish. Aquat. Sci., 1994, 51: 1～7.
37. Watahiki M, et al. cDNA cloning and primary structure of yellow tail (Seriola Quinqueradiata) pregrowth hormone. Gen. Comp, Endocrinol, 1988, 70:401.
38. Watahiki M. Conserved and unique amino acid residues in domains of the growth hormone. J. Biochem, 1989, 264: 312.
39. Zhang YZ, Basic experiments in molecular cloning, 1995.
40. Zhu Z, He L, Chen T T. Primary-structural. and evolutionary analyses of the growth hormone gene from grass carp (Ctenspharynogodom idellus). Eur. J. Biochem., 1992, 207: 643～648.
41．白俊杰，简清，马进等．鲑鱼生长激素cDNA在大肠杆菌中的表达及表达产物对罗非鱼促生长作用．农业生物技术学报，1998，6(4)：343～346．
42．白俊杰，马进，简清等．鲤鱼生长激素基因克隆及原核表达．中国生物化学与分子生物学报，1999，15(3)：409～412．
43．陈松林等．用酶联免疫吸附受体法检测鱼类生长激素的生物活性[J]．水产学报，1992，19(3)：217～224．
44．李英华，白俊杰，李新辉等．鲤鱼生长激素在毕赤酵母中的表达[J]．中国生物化学与分子生物学报，2001，17(4)：488～491
45．卢圣栋主编．现代分子生物学技术．北京：高等教育出版社，1993．381～385．
46．孙逊，朱尚权．生长激素的结构与功能[J]．国外医学生理、病理科学与临床分册，1999，19(1)：6～9
47．王伟，汪亚平，朱作言．人工合成草鱼生长激素cDNA在大肠杆菌中的表达[J]．遗传学报，2001，28(4)：306～312．
48．肖东，陈松林，严安生等．胡子鲶血浆中外源生长激素的代谢动力学[J]．水产学报，1999，23(3)：234
49．胥传来，乐国伟，姚惠源．长激素(PST)脂质体对育肥猪生产性能的影响[J]．家畜生态，2002，23(1)：36～37．
50．胥传米，乐国伟，姚惠源等．生长激素脂质体的研制[J]．精细化工 2001，18(12)：718～719．
51．许克圣，魏彦章，郭礼和等．转移人生长激素基因和注射人生长激素基对促进银鲫生长的研究[J]．水生生物学报，1991，15(2)：103～109．
52．杨丰，郑斌，徐洵．三种鱼类生长激素cDNA基因结构的比较研究[J]．生物化学与生物物理学报，1995，27(5)：537～542
53．张智清等．PRPL启动子原核表达载体的构建[J]．病毒学报，1990，6：102．
54．周宇荀，魏，尔芝，王二力，融合蛋白表达载体pGEX及其应用[J]．生命科学，1998，10(3)：122～124．
2. Ber R, Daniel V. Structure and sequence of the growth hormone-encoding gene from Tilapia nilolica. Gene, 1992, 113: 245.
3. Chen S L, Deng W T, Liu X T, et al. Isolation and purification of growth hormone from grass carp(Ctenspharynogodom idellus) and preparation of antibody against the gcGH [J]. Acta Zoologica Sinica, 1995, 41: 282～290.
4. Chen S L, Hong Y H, Schartl M. Lack of ultraviolet light inducibility of the medakafish (Oryzias latipes) tumor suppress gene p53 [J]. Gene, 2001, 264: 197～203.
5. Chen S L, Yang F, He L, et al. Generation and characterization of monoclonal antibodies against grass carp (Ctenspharynogodom idellus) growth hormone [J]. Asian Fisheries Science, 1996, 9: 183～189.
6. Chen S L. Progress and prospect of gene engineering of fish peptide hormone [J]. J Fish China, 1993, 17: 264～270.
7. Clark W C, Farmer S W, Hartwell K M. Gene. Comp. Endocrinol., 1997, 27: 174.
8. Degani G, Gallagher M L. J. Fish. Aquat. Sci., 1985, 42: 185.
9. Donaldson E M, Fagerlund U H M, Higgs D A, McBride J R. Hormonal enhancement of growth. Fish Physiol, NewYork: Academic Press, 1979, 8: 455～597.
10. Duan C, Hirano T. An guillajaponica. Aquaculture, 1991, 95: 179.
11. Fine M, Sakal E, Vashdi D, et al. Recombinant carp (Cyprinus carp) growth hormone: expression, purification and determination of biological activity in vitro and in vivo [J]. Gene Comp Endocrinol, 1993, 89: 51～61.
12. Fu C, Cui Y, Hung S S O, Zhu Z. J. Fish. Bio., 1998, 53: 115.
13. Funkenstein B, et al. Cloning and sequencing of the Glithead seabream (Sparus aurata) growth homone-encodong cDNA. Gene, 1991, 103: 343.
14. Georgopoulou U, Sire M F, Vernier J M. Immunological demon-stration of intestinal absorption and digestion of protein macro-molecules in the trout (Salmogairdneri). Cell Tissue. Res, 1986, 245: 387～395.
15. Gill J A, Sumter J P, Donaldson E M, Dye H M, Souza L M, Berg T, Wypych J, Langley K. Recombinant chicken and bovine growth hormones accelerate growth in aquacultured juvenile Pacific salmon Oncorhynchus kisutch. Biotechnology, 1985, 3: 643～646.
16. Hertz Y et al. Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus carpio) and its enhancement by deoxycholate. J. Comp. Physiol., 1991, 161B: 159～163.
17. Hew C L, Trinh K Y, Du S J, et al. Molecular cloning and expression of salmon pituitary hormone [J]. Fish Physiol Biochem, 1989, 7: 375～380.
18. Ho W K K, et al. Cloning of the grass carp growth hormone cDNA. Biochem. Biophys. Res. Commun., 1990, 161: 1239.
19. Hong Y, Schartl M. Seqence of the growth hormone gene from the silver carp and evolution of GH genes in vertebrates [J]. Biochem Biophys Acta, 1993, 1174: 285～288.
20. Koren Y, Sarid S, Ber R, Daniel V. Carp growth hormone; molecular cloning and sequencing of cDNA. Gene, 1989,77: 309～315.
21. Lebail P Y, Sire M F, Vernier J M. Intestinal transfer for growth hormone into the circulatory system of the rainbow trout Salmo gairdneri: Interference by granule cell. J. Exp. Zool, 1989, 251: 101～107.
22. Marchant T A, Peter R E, Seasonal variations in body growth rates and circulation levels of growth hormones in the gold fish, Carassius auratus [J]. J Exp. Zool, 1986, 237～239.
23. Mclean E, Donaldson E M, Dye H M, Souza L M. Growth acceleration of coho salmon (Oncorhynchus kisutch) following oral administration of recombinant bovine somatotropin. Aquaculture, 1990, 91: 197～203.
24. Mclean E, Donaldson E M. The role of growth hormone in growth of poikilotherms. In: Schriebman M P, Scanes C G, Pang P K T, eds. The Endocrinology of Growth, Development and Metabolism in Vertebrates. SanDiego: Academic Press, 1993, 43～71.
25. Miler W L, Eberhardt N L. Structure and evolution of growth hormone gene family. Endocrine Rev., 1983, 4: 97.
26. Moore D D, et al. Structure, expression and evoluticn of growth hormone genes. Rec. Proc. Horm. Res, 1982, 38: 197.
27. Moriyama S, Yamamoto H, Sugimoto S et al. Oral administration of recombinant salmon
growth hormone to rainbow trout, Oneorhynehus mykiss. Aquaeulture, 1993, 112: 99～106.
28. Rentier-Delrue F, Swennen D et al. Tilapia growth hormone: Molecular cloning of cDNA and expression in Escherichia coli. DNA, 1989, 8: 271～278.
29. Saito A, et al. Molecular cloning of eel growth hormone cDNA and its expression in Escherichia coli. Gene, 1988, 73:545.
30. Sambrook J, et al. Molecular cloning: Alaboratory Manual. 2nded, NewYork: Cold Spring Harbor Laboratory Press, 1989.
31. Sato N, et al. Molecular cloning and nucleotide sequence of tuna growth hormone Cdna. Biochem. Biophys. Acta., 1988, 949: 35.
32. Schoepfer R. The pRSET fa mily of T7 promoter expression vectors for Escherichia coli[J]. Gene, 1993, 124: 83～85.
33. Sekine S, et al., Isolation and characterization of a novel growth hormone cDNA from salmon (Oncohynchus keta). Biochem. Biophys. Acta., 1989, 1009: 117.
34. Sekine S, Mizukami T, Nishi T et al. Cloning and expressing of cDNA for salmon growth hormone in Escherichia coli. Proc. Natl. Acad. Sci. USA, 1985, 82: 4306～4310.
35. Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15; 67(1): 31-40.
36. Tsai H J, Chen H M, Lo C F. Sceretory synthesis of active recombinant fish growth hormone by insect cells using a Bsculovirus Vector. Can. J. Fish. Aquat. Sci., 1994, 51: 1～7.
37. Watahiki M, et al. cDNA cloning and primary structure of yellow tail (Seriola Quinqueradiata) pregrowth hormone. Gen. Comp, Endocrinol, 1988, 70:401.
38. Watahiki M. Conserved and unique amino acid residues in domains of the growth hormone. J. Biochem, 1989, 264: 312.
39. Zhang YZ, Basic experiments in molecular cloning, 1995.
40. Zhu Z, He L, Chen T T. Primary-structural. and evolutionary analyses of the growth hormone gene from grass carp (Ctenspharynogodom idellus). Eur. J. Biochem., 1992, 207: 643～648.
41．白俊杰，简清，马进等．鲑鱼生长激素cDNA在大肠杆菌中的表达及表达产物对罗非鱼促生长作用．农业生物技术学报，1998，6(4)：343～346．
42．白俊杰，马进，简清等．鲤鱼生长激素基因克隆及原核表达．中国生物化学与分子生物学报，1999，15(3)：409～412．
43．陈松林等．用酶联免疫吸附受体法检测鱼类生长激素的生物活性[J]．水产学报，1992，19(3)：217～224．
44．李英华，白俊杰，李新辉等．鲤鱼生长激素在毕赤酵母中的表达[J]．中国生物化学与分子生物学报，2001，17(4)：488～491
45．卢圣栋主编．现代分子生物学技术．北京：高等教育出版社，1993．381～385．
46．孙逊，朱尚权．生长激素的结构与功能[J]．国外医学生理、病理科学与临床分册，1999，19(1)：6～9
47．王伟，汪亚平，朱作言．人工合成草鱼生长激素cDNA在大肠杆菌中的表达[J]．遗传学报，2001，28(4)：306～312．
48．肖东，陈松林，严安生等．胡子鲶血浆中外源生长激素的代谢动力学[J]．水产学报，1999，23(3)：234
49．胥传来，乐国伟，姚惠源．长激素(PST)脂质体对育肥猪生产性能的影响[J]．家畜生态，2002，23(1)：36～37．
50．胥传米，乐国伟，姚惠源等．生长激素脂质体的研制[J]．精细化工 2001，18(12)：718～719．
51．许克圣，魏彦章，郭礼和等．转移人生长激素基因和注射人生长激素基对促进银鲫生长的研究[J]．水生生物学报，1991，15(2)：103～109．
52．杨丰，郑斌，徐洵．三种鱼类生长激素cDNA基因结构的比较研究[J]．生物化学与生物物理学报，1995，27(5)：537～542
53．张智清等．PRPL启动子原核表达载体的构建[J]．病毒学报，1990，6：102．
54．周宇荀，魏，尔芝，王二力，融合蛋白表达载体pGEX及其应用[J]．生命科学，1998，10(3)：122～124．