家蚕30kD载脂蛋白19G1的表达和功能的初步研究
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摘要
30K蛋白家族是一类在氨基酸组成及免疫学活性上相似的蛋白,分子量约为30 kD~([1,2])。家蚕30K蛋白是由脂肪体合成的非雌特异性脂蛋白,表达受保幼激素的调节,是生长发育过程中主要储藏蛋白之一,是五龄幼虫及蛹主要的蛋白组分,主要分布在血淋巴中~([3])。对雌性家蚕,30K蛋白还是卵黄原蛋白的重要组成部分~([3])。30K蛋白中研究比较多的是30 kD载脂蛋白6G1和19G1。Kim EJ~([1])与Park HJ~([4])分别把家蚕30Kc6基因构建入表达载体在大肠杆菌中表达得到重组蛋白,纯化后添加到哺乳动物及昆虫细胞培养基中,能抑制哺乳动物及昆虫细胞的凋亡;Koo TY~([5])等构建了表达重组30Kc6基因的生产干扰素的CHO细胞系,结果表明30Kc6基因的表达抑制细胞的凋亡并提高干扰素的产量。
本研究从家蚕蛹cDNA文库中通过生物信息学分析发现一条低分子量30 kD载脂蛋白19G1前体(low molecular mass 30 kD lipoprotein 19G1 precursor)的EST序列(GenBank登录号:AY568957)。生物信息学分析结果表明该基因ORF不含内含子,由长为771 bp的单一外显子编码256个氨基酸残基的蛋白质,预测分子量约为29.5 kD,等点电为7.290。我们将其ORF克隆到原核表达载体pET-28a(+)中,构建重组质粒,双酶切鉴定及序列测定正确后转化大肠杆菌BL21(DE3),经IPTG诱导表达的重组蛋白以包涵体形式存在,经过镍柱亲和层析纯化后获得高纯度的目的蛋白,以该重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体,经ELISA检测,抗体效价可达1:12800,Western blotting结果显示抗体的特异性较好。
提取家蚕各个时期和五龄幼虫各组织的总RNA分别进行荧光定量PCR分析结果表明,家蚕发育时期在蛹期转录水平最高,卵中最低;五龄幼虫时期在脂肪体中转录水平较高,丝腺和肠较低。提取家蚕各发育时期以及家蚕五龄幼虫各组织蛋白进行Western blotting分析,结果表明,该蛋白在家蚕蛹中及五龄幼虫的脂肪体、卵巢表达较高。
把纯化的蛋白添加到家蚕Bm5细胞培养基中,结果表明重组30 kD载脂蛋白19G1能抑制化学试剂(地塞米松)诱导的家蚕Bm5细胞的凋亡。
The 30K protein group have common characteristics in amino acid composition and immunological activity. They are a group of structurally related proteins with a molecular mass of approximately 30 kD. 30K proteins are not female specific lipoproteins, which are synthesized in the fat body of silkworm. Furthermore, it was found that expression of these lipoproteins is regulated by juvenile hormone. They are the main storage proteins used for growth of silkworm and the major protein components of the hemolymph of the fifth instar larvae and of pupae. In female, 30K proteins are the major component of the yolk protein. Among the 30K protein, there are more researches on 30 kD lipoprotein 6G1 and 19G1. The 30Kc6 gene was cloned into expression vector to produce this protein in Escherichia coli, and the protein was purified. The purified 30K protein effectively inhibited insect cell and mammalian cell apoptosis by addition to culture medium. An interferon-b producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed. The results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.
The EST of low molecular mass 30 kD lipoprotein 19G1 precursor (GenBank accession no. AY568957) was found through searching our cDNA bank of silkworm pupae. By means of bioinformatic methods, the ORF contains no intron but an exon of 771 bp encoding 256 amino acids; the predicted molecular weight is 29.5 kD and the pI is 7.290. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a(+) and the recombinant plasmid was transformed into E.coli BL21(DE3). The fusion protein was successfully expressed,purified and then immuned New Zealand rabbit to get antiserum. The titer of the polyclonal antibodies reaches 1:12800 measured by ELISA. We compared the quantity of 30Kc19 mRNA in different tissues of the fifth instar larva and different stages of silkworm using RT-PCR. And we demonstrated that apoptosis is inhibited by supplementing the culture medium with this purified recombinant 30K protein.
本研究从家蚕蛹cDNA文库中通过生物信息学分析发现一条低分子量30 kD载脂蛋白19G1前体(low molecular mass 30 kD lipoprotein 19G1 precursor)的EST序列(GenBank登录号:AY568957)。生物信息学分析结果表明该基因ORF不含内含子,由长为771 bp的单一外显子编码256个氨基酸残基的蛋白质,预测分子量约为29.5 kD,等点电为7.290。我们将其ORF克隆到原核表达载体pET-28a(+)中,构建重组质粒,双酶切鉴定及序列测定正确后转化大肠杆菌BL21(DE3),经IPTG诱导表达的重组蛋白以包涵体形式存在,经过镍柱亲和层析纯化后获得高纯度的目的蛋白,以该重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体,经ELISA检测,抗体效价可达1:12800,Western blotting结果显示抗体的特异性较好。
提取家蚕各个时期和五龄幼虫各组织的总RNA分别进行荧光定量PCR分析结果表明,家蚕发育时期在蛹期转录水平最高,卵中最低;五龄幼虫时期在脂肪体中转录水平较高,丝腺和肠较低。提取家蚕各发育时期以及家蚕五龄幼虫各组织蛋白进行Western blotting分析,结果表明,该蛋白在家蚕蛹中及五龄幼虫的脂肪体、卵巢表达较高。
把纯化的蛋白添加到家蚕Bm5细胞培养基中,结果表明重组30 kD载脂蛋白19G1能抑制化学试剂(地塞米松)诱导的家蚕Bm5细胞的凋亡。
The 30K protein group have common characteristics in amino acid composition and immunological activity. They are a group of structurally related proteins with a molecular mass of approximately 30 kD. 30K proteins are not female specific lipoproteins, which are synthesized in the fat body of silkworm. Furthermore, it was found that expression of these lipoproteins is regulated by juvenile hormone. They are the main storage proteins used for growth of silkworm and the major protein components of the hemolymph of the fifth instar larvae and of pupae. In female, 30K proteins are the major component of the yolk protein. Among the 30K protein, there are more researches on 30 kD lipoprotein 6G1 and 19G1. The 30Kc6 gene was cloned into expression vector to produce this protein in Escherichia coli, and the protein was purified. The purified 30K protein effectively inhibited insect cell and mammalian cell apoptosis by addition to culture medium. An interferon-b producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed. The results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.
The EST of low molecular mass 30 kD lipoprotein 19G1 precursor (GenBank accession no. AY568957) was found through searching our cDNA bank of silkworm pupae. By means of bioinformatic methods, the ORF contains no intron but an exon of 771 bp encoding 256 amino acids; the predicted molecular weight is 29.5 kD and the pI is 7.290. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a(+) and the recombinant plasmid was transformed into E.coli BL21(DE3). The fusion protein was successfully expressed,purified and then immuned New Zealand rabbit to get antiserum. The titer of the polyclonal antibodies reaches 1:12800 measured by ELISA. We compared the quantity of 30Kc19 mRNA in different tissues of the fifth instar larva and different stages of silkworm using RT-PCR. And we demonstrated that apoptosis is inhibited by supplementing the culture medium with this purified recombinant 30K protein.
引文
[1] Kim EJ, Park HJ, Park TH. Inhibition of apoptosis by recombinant 30K protein originating from silkworm hemolymph[J]. Biochem.Biophys. Res.Commun, 2003, 308(3): 523-528.
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[4] Park HJ, Kim EJ, Koo TY, et al. Purification of recombinant 30K protein produced in Escherichia coli and its anti-apoptotic effect in mammalian and insect cell systems[J]. Enzyme Microbial.Technol,2003,33(4): 466-471.
[5] Koo TY,et al. Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-b in serum-free suspension culture of CHO cells[J]. Process Biochem, (2008), doi:10.1016/j.procbio.2008.09.018
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[3] Minoru U, Akihiri K, Daisuke N, et al. Specific binding of silkworm Bombyx mori 30-kD liproproteins to carbohydrates containing glucose[J]. Biosci.Biochnol.Biochem, 2002, 66(10): 2264-2266.
[4] Park HJ, Kim EJ, Koo TY, et al. Purification of recombinant 30K protein produced in Escherichia coli and its anti-apoptotic effect in mammalian and insect cell systems[J]. Enzyme Microbial.Technol,2003,33(4): 466-471.
[5] Koo TY,et al. Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-b in serum-free suspension culture of CHO cells[J]. Process Biochem, (2008), doi:10.1016/j.procbio.2008.09.018
[6] Rhee WJ, Kim EJ, Park TH. Kinetic effect of silkworm hemolymph on the delayed host cell death in an insect cell–baculovirus system[J]. Biotechnol Prog, 1999, 15(6): 1028-1032.
[7] Rhee WJ, Kim EJ, Park TH. Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells[J]. Biochem.Biophsy.Res.Commun, 2002, 295(4): 779-783.
[8] Kim EJ, Rhee WJ, Park TH. Isolation and characterization of an apoptosis-inhibiting component from the hemolymph of Bombyx mori[J].Biochem Biophys Res Commun, 2001, 285(2): 224-228.
[9] Mori S, Izumi S, and Tomino S. Complete nucleotide sequence of major plasma protein genes of Bombyx mori[J]. Biochim.Biophys.Acta, 1991, 1090(1): 129-132.
[10]孙全,赵萍,夏庆友.家蚕30K蛋白基因组分析及蛋白质鉴定[D].重庆市北碚区天生路2号,西南大学,2008
[11] Maachupalli-Reddy J, Kelley BD, De Bernardez Clark E. Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme [J]. Biotechnol Prog, 1997, 13(2): 144-150.
[12] Tojo S, Betchaku T, Ziccardi VJ, et a1. Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia[J]. J Cell Biol, 1978, 78: 823-838.
[13] Mine E, Izumi S, Katsuki M, et a1. Development and sex.dependent regulation of storage protein synthesis in the silkworm Bombyx mori[J]. Dcv Biol, 1983, 97: 329-337.
[14] Mori S, Izumi S, Tomino S. Structures and organization of major plasma protein genes of the silkworm Bombyx mori[J].J Mol Biol, 1991, 218: 7-12.
[15]唐平,王文兵,李兵等.利用杆状病毒系统在昆虫细胞TN5中表达家蚕30K蛋白[J].蚕业科学,2006,32(2):169-173.
[16] Minoru U, Yosuke K, Ichiro K, et al. Glucan-binding activity of silkworm 30-kD apolipoprotein and its involvement indefense against fungal infection[J]. Biosci. Biochnol, Biochem, 2005,69(6): 1178-1185.
[17] Msjno G, Joris I. Apoptosis. Oncosis and necrosis: an overview of cell death[J]. Am J Pathol, 1995. 1469(1): 3-15.
[18] Jtattel M. Escaping cell death: survival proteins in cancer[J]. Exp Cell Res, 1999, 248: 30-43.
[19] Kim EJ, Park TH. Anti-apoptosis engineering[J]. Biotechnol Bioprocess Eng, 2003, 8: 76-82.
[20]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. 547-580.
[21]唐平,李兵,沈卫德等. 30K蛋白对家蚕及幼虫存活的作用[J].昆虫知识,2007, 44(6):826-829.
[22] Sakai K, Matsunaga T, Hayashi C, Yamaji H, Fukuda H. Effect of phosphatidic acid on recombinantprotein production by Chinese hamster ovary cells in serum-free culture[J]. Biochem Eng J, 2002, 10: 85-92.
[23] Kallel H, Jouini A, Majoul S, Rourou S. Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells[J]. Biotechno,l 2002, 95: 195-204.
[24] Ryu JH, Kim SS, Cho SW, Choi CY, Kim BS. HEK 293 cell suspension culture using fibronectin-adsorbed polymer nanospheres in serum-free medium[J]. Biomed Mater Res A, 2004, 71: 128-133.
[25] Kim EJ, Rhee WJ, Park TH. Inhibition of apoptosis by a bombyx mori gene[J]. Biotechnol Prog, 2004, 20: 324-329.
[26] Choi SS, Rhee WJ, Park TH. Inhibition of human cell apoptosis by silkworm hemolymph[J]. Biotechnol Prog, 2002, 18: 874-878.
[27] Choi SS, Rhee WJ, Park TH. Beneficial effect of silkworm hemolymph on a CHO cell system: inhibition of apoptosis and increase of EPO production[J]. Biotechnol Bioeng, 2005, 91: 793-800.
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