猪α型干扰素基因的克隆及其在大肠杆菌中的表达
摘要
干扰素(IFN)是一类重要的细胞因子，具种属特异性，在同种细胞上具有广谱的抗病毒、抗细胞增殖、免疫调节等多种生物活性。其中IFN-α／β是机体的第一抵御病毒体系。猪是一种重要的经济动物，利用重组DNA技术生产有广泛抗病毒效应的基因工程猪α型干扰素(PoIFN-α)无疑有重要意义。
本研究从猪肝细胞中提取基因组DNA，运用常规PCR方法扩增得到一个内部含终止子的假基因，登录Genebank号为AF350425。比较此假基因序列和PoIFNα1基因序列的差别，结合AS-PCR方法及SOE技术获得了猪α型干扰素基因的成熟肽编码区。所得序列记为PoIFN-α1a，与PoIFN-α1基因成熟肽编码区相比有5个碱基差别，导致3个氨基酸不同。
首先以非融合蛋白形式进行表达。将PoIFN-α1a插入到原核表达载体pGRW，转化大肠杆菌DH5α，得到的重组菌株经诱导有以包涵体形式表达的目的蛋白条带出现，表达量约10％。提取包涵体，变、复性后，以WISH／VSV系统检测其抗病毒活性约为3200IU／mg。而采用巨引物PCR法介导的定点突变将PoIFN-α1a第86位的Cys突变为Tyr，同时将N端首位氨基酸(Cys)的密码子TGT同义突变为大肠杆菌偏爱的密码子TGC，突变后基因记作PoIFN-α1b。将其与融合蛋白表达载体pGEX-4T-3连接，转化大肠杆菌BL-21，诱导后表达产物占菌体总蛋白的20％。将以包涵体形式表达的目的蛋白经变、复性处理，并以FPLC进一步纯化，得到产物在WISH／VSV系统上的抗病毒活性为5200IU／mg。
Interferons (IFNs), a family of cytokines, have many kinds of biological activities, such as interfering with the replication of various viruses, decreasing cell proliferation and modifying immunological processes. IFN- a / P is the first system of body to defend virus attacking. Pig production is important for food industry and the development of recombant porcine interferon- a ( rPoIFN- a ) is strongly desirable for prevention of pig virus diseases.
A pseudogene with a termination codon TAG in the middle was isolated from porcine liver by PCR method. The sequence had been send to Genebank and the accession number was AF350425. Then a PoIFN-a gene was produced by AS-PCR and SOE using four special primers based on the differences between the sequence of the pseudogene and PoIFN- Q 1. hi the sequence of the PoIFN- a gene five nucleotides were different from the sequence of PoIFN-a i, which leaded to three amino acids difference.
To obtain a recombinant mature PoIFN- a , plasmid pGRW-PoIFN- a 1 a was constructed. The expression of the unfused protein in the strain E. coli DH5 a was 10%, and the antiviral activity of the protein was 3200IU/mg. Then a site-directed mutation of Cys(86) to Tyr was made by megaprimer PCR ; meanwhile TGT, the first codon of PoIFN- a , was changed to TGC , which was a bias codon to E. coli. In order to increase the expression level of PoIFN- a , the mutated sequence was inserted into the fused protein expression plasmid pGEX-4T-3. The expressed fused protein, which was found in insoluble form, counted for 20% of the total cell proteins. After denaruration, renaturation and purification, a higher antiviral activity was measured (5200IU/mg).
本研究从猪肝细胞中提取基因组DNA，运用常规PCR方法扩增得到一个内部含终止子的假基因，登录Genebank号为AF350425。比较此假基因序列和PoIFNα1基因序列的差别，结合AS-PCR方法及SOE技术获得了猪α型干扰素基因的成熟肽编码区。所得序列记为PoIFN-α1a，与PoIFN-α1基因成熟肽编码区相比有5个碱基差别，导致3个氨基酸不同。
首先以非融合蛋白形式进行表达。将PoIFN-α1a插入到原核表达载体pGRW，转化大肠杆菌DH5α，得到的重组菌株经诱导有以包涵体形式表达的目的蛋白条带出现，表达量约10％。提取包涵体，变、复性后，以WISH／VSV系统检测其抗病毒活性约为3200IU／mg。而采用巨引物PCR法介导的定点突变将PoIFN-α1a第86位的Cys突变为Tyr，同时将N端首位氨基酸(Cys)的密码子TGT同义突变为大肠杆菌偏爱的密码子TGC，突变后基因记作PoIFN-α1b。将其与融合蛋白表达载体pGEX-4T-3连接，转化大肠杆菌BL-21，诱导后表达产物占菌体总蛋白的20％。将以包涵体形式表达的目的蛋白经变、复性处理，并以FPLC进一步纯化，得到产物在WISH／VSV系统上的抗病毒活性为5200IU／mg。
Interferons (IFNs), a family of cytokines, have many kinds of biological activities, such as interfering with the replication of various viruses, decreasing cell proliferation and modifying immunological processes. IFN- a / P is the first system of body to defend virus attacking. Pig production is important for food industry and the development of recombant porcine interferon- a ( rPoIFN- a ) is strongly desirable for prevention of pig virus diseases.
A pseudogene with a termination codon TAG in the middle was isolated from porcine liver by PCR method. The sequence had been send to Genebank and the accession number was AF350425. Then a PoIFN-a gene was produced by AS-PCR and SOE using four special primers based on the differences between the sequence of the pseudogene and PoIFN- Q 1. hi the sequence of the PoIFN- a gene five nucleotides were different from the sequence of PoIFN-a i, which leaded to three amino acids difference.
To obtain a recombinant mature PoIFN- a , plasmid pGRW-PoIFN- a 1 a was constructed. The expression of the unfused protein in the strain E. coli DH5 a was 10%, and the antiviral activity of the protein was 3200IU/mg. Then a site-directed mutation of Cys(86) to Tyr was made by megaprimer PCR ; meanwhile TGT, the first codon of PoIFN- a , was changed to TGC , which was a bias codon to E. coli. In order to increase the expression level of PoIFN- a , the mutated sequence was inserted into the fused protein expression plasmid pGEX-4T-3. The expressed fused protein, which was found in insoluble form, counted for 20% of the total cell proteins. After denaruration, renaturation and purification, a higher antiviral activity was measured (5200IU/mg).
引文
1．徐耀先，周晓峰，刘立德，分子病毒学，湖北科技技术出版社，2000，1，175～185．
2．侯云德，分子病毒学，北京：文苑出版社，1990，237～246．
3. Lefevre F, Bonnardiere C L, Molecular Cloning and Sequencing of a Gene Encoding Biologically Active Porcine α -Interferon. J Interferon Res, 1986, 6, 349～360.
4. Peter L, Biochemistry of Interferons and Their Actions, Ann Rev Biochem, 1982, 51:251～282.
5. Pestka S, Langer J A, et al. Interferons and their actions, Ann Rev Biochem, 1987, 56, 727～777.
6．王伟，侯云德，人α型干扰素突变体(IFN-α1/86D)的组建及其生物活性的研究，病毒学报，1990，6(4)，322～325．
7. George R S, Ian M K, Bryan R G, et al, How Cells Respond to Interferons, Ann Rev Biochem, 1998, 67, 227～264.
8．罗经，杨学楼，猪干扰素的某些性质，国外医学：免疫学分册，12(5)，252-255．
9. Egbert P, Properties of Natural Porcine Interferons, J Interferon Res, 1988, 8, 61～73.
10. Lefevre F, L'haridon R, Borras-Cuesta F, et al. Production, purification and biological properties of an Escherichia coli-derived recombinant porcine alpha interferon, J Gen Vir, 1990, 71, 1057～1063.
11．郭赢军，吴丹，陈蕊雯，猪干扰素在大肠杆菌中的克隆与表达，生物工程学报，2001，17(2)，183～186．
12．夏春，刘津，杨琪等，猪干扰素β基因的分子克隆与测序，中国兽医杂志，2000，26(6)，6～7．
13．夏春，汪明，蒋金书等，猪干扰素IFN与IFN基因克隆及其生物发酵体系，大连，中国畜牧兽医学会生物技术学分会、中国免疫学会兽医免疫学分会论文集，2000年．
14．Moore B.R, 干扰素在大家畜临床上的应用，国外兽医学—畜禽传染病，1997，73(3)，18～22．
15．陈国华，朱耀华，邓秀兰等，干扰素对猪腹泻病的治疗实验．上海畜牧兽医通讯，1998，3，22．
16．刘万均，胡景韶，张道永等，用猪白细胞干扰素防治猪流行性腹泻，中国兽医科技，1996，26(2)，27～28．
17．丁敦志，李品齐，王劲等，猪白细胞干扰素诱生条件的研究，四川畜牧兽医，1996，3(1)，20～21．
18．杨歧生，分子生物学基础，杭州：浙江大学出版社，1998，42～43．
19. Bottema C, PCR amplification of specific alleles. Mut Res, 1993, 288(1): 93～102.
20．C.W.迪芬巴赫，G.S.德维克斯勒，PCR技术实验指南，北京：北京科学出版社，1998，431～436．
21. Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual; second edition, cold spring harbor laboratory. Cold spring harbor, NY, 1989, 26～27.
22. David V, Hans W, Charles W, Is Sequence Conservation in Interferons due to Selection for Functional Proteins, Nature, 1985, 698～700.
23. Fedor E R, Nikolai N K, Igor I V, Intermolecular homologies of Human Interferon-Alpha, 1992,186, (1), 211～218.
24. Jaap B, Kor V A, Arnold C P, et al, The Biological Activity of Interferon Alpha is Influenced by Two Distinct Regions in The Protein, Biochem and Biophysical Res Commu, 1989,164 (1), 22～29.
25．隋广超，胡美浩，大肠杆菌中包涵体的形成及其活性表达产物的分离，1993，3(2)，6～9
26．龙建银，王会信，重组蛋白的体外再折叠，生理科学进展，1998，29(2)，103～108．
27．纪剑飞，张成刚，包涵体重组蛋白的纯化及复性，沈阳药科大学学报，1998，15(4)，303～307．
28．张毅，屈贤铭，陆坚峰等，重组GM-CSF/IL-3融合蛋白的纯化，生物化学与生物物理学报，2000，32(3)，235～238．
29. Marston F A O, Freedman R B, Recovery and Reactivation of Recombinant Proteins, Biochem Society Transactions, 1988, 16, 101～104.
30. Bernhard E F, Renaturation of Recombinant Proteins Produced as Inclusion Bodies, Biotech Adv, 1994, 12, 89～101.
31. Bernhard F, Ian S, Peter G, Isolation, Renaturation, and Formation of Disulfide Bonds of Eukaryotic Proteins Expressed in as Inclusion Bodies, Biotec and Bioengineering, 1993, 41, 3～13.
32. Langley K E, Berg T F, Strickland T W, et al. Recombinant-DNA-derived Bovine Growth Hormone from Escherichia coli, Eur. J Biochem, 1987, 163, 313～321.
33. Doonan S, Walker J M, Downstream Pocessing of Protein Products, Biochem Society Transactions, 1990, 18, 231～233.
34. David N B, Scott M P, Henry A H, et al, Equilibrium Denaturation of Pituitary- and Recombinat-Derived Bovine Growth Hormone, American Chem Soc, 1985, 24, 7662～7668.
35. Sung C L, Kai L L, Hsin C C, Enhanced Protein Renatration by Temperature-Responsive Polymers, Biotechnology and bioengineering, 2000, 67(5), 505～511.
36．焦建伟，俞梅敏，茹炳根，真核低分子量尿激酶原突变体基因在大肠杆菌中的高效表达，中国生物化学与分子生物学报，2001，17(3)，340～343．
37．史晋辉，董晓燕，孙彦，盐酸胍浓度对变性溶菌酶复性的影响，生物化学与生物物理学报，2001，33(4)，447～451．
38. Michael C, Nirdosh K P, Malcolm R B, Comparing the Refolding and Reoxidation of Recombinant Porcine Growth Hormone from a Urea Denatured State and from Escherichia coli Inclusion Bodies, Biochemistry, 1995, 34(17), 5773～5794.
39. Marshak D, Kadonaga J T, Burgess R R, Strategies for protein purification and characterization: A Laboratory course Manual Cold Spring Harbor Laboratory Press, 1996.
40．丁敦志，蒋开全，李品齐，猪白细胞干扰素效价测定方法的探讨，四川畜牧兽医，1996，4，20～21．
41．孙卫民，王惠琴，细胞因子研究方法学，北京：人民卫生出版社，1999，540～561．
42．李江，魏开坤，马学军等，新型人干扰素a1c的纯化和活性测定，细胞与分子免疫学杂志，2001，17(1)，79～81．
43. Ernest K J, Diana F, Human Fibroblast Interferon, J Bio Chem, 1981, 256(8), 3609～3611.
44．李燕，侯云德，人型干扰素86位单—氨基酸置换可以改变其生物学活性，中华微生物学和免疫学杂志，1990，10(4)，248～251．
45．王翔，俞伟源，缺失突变法研究尿激酶-单链抗体融合基因在大肠杆菌中的表达，1999，15(1)，23～27．
46. Sarker G, Sommer S S, The "megaprimer" method of site-directed mutagenesis, Bio Tech, 1990, 8, 404～407.
47．张学敏，王宜强，靶向新基因的分子克隆策略—理论与方法，军事医学科学出版社．1999，82～85．
2．侯云德，分子病毒学，北京：文苑出版社，1990，237～246．
3. Lefevre F, Bonnardiere C L, Molecular Cloning and Sequencing of a Gene Encoding Biologically Active Porcine α -Interferon. J Interferon Res, 1986, 6, 349～360.
4. Peter L, Biochemistry of Interferons and Their Actions, Ann Rev Biochem, 1982, 51:251～282.
5. Pestka S, Langer J A, et al. Interferons and their actions, Ann Rev Biochem, 1987, 56, 727～777.
6．王伟，侯云德，人α型干扰素突变体(IFN-α1/86D)的组建及其生物活性的研究，病毒学报，1990，6(4)，322～325．
7. George R S, Ian M K, Bryan R G, et al, How Cells Respond to Interferons, Ann Rev Biochem, 1998, 67, 227～264.
8．罗经，杨学楼，猪干扰素的某些性质，国外医学：免疫学分册，12(5)，252-255．
9. Egbert P, Properties of Natural Porcine Interferons, J Interferon Res, 1988, 8, 61～73.
10. Lefevre F, L'haridon R, Borras-Cuesta F, et al. Production, purification and biological properties of an Escherichia coli-derived recombinant porcine alpha interferon, J Gen Vir, 1990, 71, 1057～1063.
11．郭赢军，吴丹，陈蕊雯，猪干扰素在大肠杆菌中的克隆与表达，生物工程学报，2001，17(2)，183～186．
12．夏春，刘津，杨琪等，猪干扰素β基因的分子克隆与测序，中国兽医杂志，2000，26(6)，6～7．
13．夏春，汪明，蒋金书等，猪干扰素IFN与IFN基因克隆及其生物发酵体系，大连，中国畜牧兽医学会生物技术学分会、中国免疫学会兽医免疫学分会论文集，2000年．
14．Moore B.R, 干扰素在大家畜临床上的应用，国外兽医学—畜禽传染病，1997，73(3)，18～22．
15．陈国华，朱耀华，邓秀兰等，干扰素对猪腹泻病的治疗实验．上海畜牧兽医通讯，1998，3，22．
16．刘万均，胡景韶，张道永等，用猪白细胞干扰素防治猪流行性腹泻，中国兽医科技，1996，26(2)，27～28．
17．丁敦志，李品齐，王劲等，猪白细胞干扰素诱生条件的研究，四川畜牧兽医，1996，3(1)，20～21．
18．杨歧生，分子生物学基础，杭州：浙江大学出版社，1998，42～43．
19. Bottema C, PCR amplification of specific alleles. Mut Res, 1993, 288(1): 93～102.
20．C.W.迪芬巴赫，G.S.德维克斯勒，PCR技术实验指南，北京：北京科学出版社，1998，431～436．
21. Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual; second edition, cold spring harbor laboratory. Cold spring harbor, NY, 1989, 26～27.
22. David V, Hans W, Charles W, Is Sequence Conservation in Interferons due to Selection for Functional Proteins, Nature, 1985, 698～700.
23. Fedor E R, Nikolai N K, Igor I V, Intermolecular homologies of Human Interferon-Alpha, 1992,186, (1), 211～218.
24. Jaap B, Kor V A, Arnold C P, et al, The Biological Activity of Interferon Alpha is Influenced by Two Distinct Regions in The Protein, Biochem and Biophysical Res Commu, 1989,164 (1), 22～29.
25．隋广超，胡美浩，大肠杆菌中包涵体的形成及其活性表达产物的分离，1993，3(2)，6～9
26．龙建银，王会信，重组蛋白的体外再折叠，生理科学进展，1998，29(2)，103～108．
27．纪剑飞，张成刚，包涵体重组蛋白的纯化及复性，沈阳药科大学学报，1998，15(4)，303～307．
28．张毅，屈贤铭，陆坚峰等，重组GM-CSF/IL-3融合蛋白的纯化，生物化学与生物物理学报，2000，32(3)，235～238．
29. Marston F A O, Freedman R B, Recovery and Reactivation of Recombinant Proteins, Biochem Society Transactions, 1988, 16, 101～104.
30. Bernhard E F, Renaturation of Recombinant Proteins Produced as Inclusion Bodies, Biotech Adv, 1994, 12, 89～101.
31. Bernhard F, Ian S, Peter G, Isolation, Renaturation, and Formation of Disulfide Bonds of Eukaryotic Proteins Expressed in as Inclusion Bodies, Biotec and Bioengineering, 1993, 41, 3～13.
32. Langley K E, Berg T F, Strickland T W, et al. Recombinant-DNA-derived Bovine Growth Hormone from Escherichia coli, Eur. J Biochem, 1987, 163, 313～321.
33. Doonan S, Walker J M, Downstream Pocessing of Protein Products, Biochem Society Transactions, 1990, 18, 231～233.
34. David N B, Scott M P, Henry A H, et al, Equilibrium Denaturation of Pituitary- and Recombinat-Derived Bovine Growth Hormone, American Chem Soc, 1985, 24, 7662～7668.
35. Sung C L, Kai L L, Hsin C C, Enhanced Protein Renatration by Temperature-Responsive Polymers, Biotechnology and bioengineering, 2000, 67(5), 505～511.
36．焦建伟，俞梅敏，茹炳根，真核低分子量尿激酶原突变体基因在大肠杆菌中的高效表达，中国生物化学与分子生物学报，2001，17(3)，340～343．
37．史晋辉，董晓燕，孙彦，盐酸胍浓度对变性溶菌酶复性的影响，生物化学与生物物理学报，2001，33(4)，447～451．
38. Michael C, Nirdosh K P, Malcolm R B, Comparing the Refolding and Reoxidation of Recombinant Porcine Growth Hormone from a Urea Denatured State and from Escherichia coli Inclusion Bodies, Biochemistry, 1995, 34(17), 5773～5794.
39. Marshak D, Kadonaga J T, Burgess R R, Strategies for protein purification and characterization: A Laboratory course Manual Cold Spring Harbor Laboratory Press, 1996.
40．丁敦志，蒋开全，李品齐，猪白细胞干扰素效价测定方法的探讨，四川畜牧兽医，1996，4，20～21．
41．孙卫民，王惠琴，细胞因子研究方法学，北京：人民卫生出版社，1999，540～561．
42．李江，魏开坤，马学军等，新型人干扰素a1c的纯化和活性测定，细胞与分子免疫学杂志，2001，17(1)，79～81．
43. Ernest K J, Diana F, Human Fibroblast Interferon, J Bio Chem, 1981, 256(8), 3609～3611.
44．李燕，侯云德，人型干扰素86位单—氨基酸置换可以改变其生物学活性，中华微生物学和免疫学杂志，1990，10(4)，248～251．
45．王翔，俞伟源，缺失突变法研究尿激酶-单链抗体融合基因在大肠杆菌中的表达，1999，15(1)，23～27．
46. Sarker G, Sommer S S, The "megaprimer" method of site-directed mutagenesis, Bio Tech, 1990, 8, 404～407.
47．张学敏，王宜强，靶向新基因的分子克隆策略—理论与方法，军事医学科学出版社．1999，82～85．