猪生长激素基因在杆状病毒载体系统中的表达
作者：欧阳菁 魏平华 杨林 谢明权 杨洁 龙綮新 王珣章
单位：欧阳菁 杨林 杨洁 龙綮新 王珣章(中山大学生物医药中心、生物防治国家重点实验室，广州 510275)；魏平华 谢明权(广东省农业科学院，广州 510650)
关键词：猪生长激素基因；序列分析；杆状病毒载体系统；表达
中国病毒学000313摘要：通过对猪生长激素(pGH)基因的cDNA进行测序，得到pGH cDNA的全序列，并与Seeburg等报道的序列进行了比较和讨论。然后利用具人工合成启动子和多角体蛋白XIV启动子的转移载体质粒pSXIVVI+X3/4构建出含pGH基因的重组质粒pX3/4-pGH。将pX3/4-pGH与致死缺失型线性化AcMNPV-OCC- DNA共转染Sf9细胞，构建出既能形成多角体又能表达pGH基因的苜蓿丫纹夜蛾核多角体重组病毒AcMNPV-pX3/4-pGH-OCC+。感染重组毒株的Hi 5细胞可溶蛋白及其培养上清的SDS-PAGE和Western blot的分析结果表明，感染细胞的蛋白电泳带的20.7 kDa处有一条猪生长激素特异带，但培养上清中没有。凝胶黑度扫描估测结果显示pGH蛋白占细胞可溶蛋白的4.48%。
, 百拇医药
中图分类号：Q786， S859.793 文献标识码：A
文章编号：1003-5125(2000)03-0285-06
Expression of Porcine Growth Hormone Gene
in Baculovirus Vector System
OUYANG Jing，YANG Lin，YANG Jie，LONG Qing-xin，WANG Xun-zhang
(Biopharmaceutical Center, State Key Laboratory for Biological Control,Zhongshan University, Guangzhou 510275, China)
, 百拇医药
WEI Ping-hua，XIE Ming-quan
(Guangdong Academy of Agricultural Sciences, Guangzhou 510650, China)
Abstract: The cDNA sequence encoding porcine growth hormone (pGH) was inserted into the plasmid of pBluescript M13(-). The sequening results showed that a 984 base-pair fragment is identical to that reported by Seeburg et al., with the exception of one different base-pair in the ORF. Then, a baculovirus recombinant vector (pX3/4-pGH) with the pGH ORF was constructed and cotransfected Hi5 cells with the linearized parental virus (AcMNPV-OCC-) DNA to produce the recombinant virus AcMNPV-pX3/4-pGH-OCC+, in which the pGH gene was driven by the synthetic and XIV promoters. The SDS-PAGE and Western blot analysis showed that the pGH gene product, 20.7 kDa fusion protein, was obtained. The expressed pGH accumulated up to about 4.48% of the soluble cellular proteins as estimated by CS-930 scanning of the SDS-PAGE gel stainning by Coomassie Brilliant Blue R250.
, http://www.100md.com
Key words: Porcine growth hormone; Sequence analysis; Baculovirus vector system; Expression
基金项目：广东省自然科学基金重点项目(963007)
作者简介：欧阳菁(1975年-)，女，湖南省人，硕士研究生，研究方向为病毒基因的调控
参考文献
[1] 苏悌之，Oxender D L, R1-Geweley M E，等.猪生长激素cDNA重组及在大肠杆菌中表达[J].生物化学与生物物理学报，1990，22(2)：111-120
[2] 余旭平，齐顺章.在大肠杆菌中温度诱导高效表达猪生长激素[J].生物工程学报，1991，7(4)：307-311
, 百拇医药
[3] O'Reilly D R, Passarelli A L, Goldman I F， et al. Characterization of the DA26 gene in a hypervariable region of the Autographa califormica nuclear polyhedrosis virus genome [J]. Journal of General Virology, 1990,71:1029-1037
[4] O'Reily D R, Miller L K, Luckow V A. Baculovirus expression vetors, A laboratory Mannul [M]. New york: WH Freeman, 1994
[5] Maeda S, Kawai T, Obinata M, et al. Production of human α-inferferon in silkworm using a baculovirus vector [J]. Natrue, 1985,315:592-594
, 百拇医药
[6] 王NFDB4章，谢伟东，龙綮新，等.形成多角体的杆状病毒载体系统的建立[J].病毒学报，1991，7(3)：253-261
[7] Sambrook J, Pritsch E F, Maniatis T, Molecular Cloning: A Laboratory Manual [M]. 2nd Ed, Cold Spring Harbor: Cold Spring Harbor Press, 1989.
[8] Summers M D, Smith G E. A manual of methods for baculovirus vectors and insect cell culture rocedures, Texas Agricultural Experiment Station Bulletin [J]. 1985,(555):27-30
[9] Seeburg P H, Sias S, Adelman J, et al. Efficient bacterial expression of bovine and porcine growth hormone [J]. DNA, 1983,2:37-45
[10] George H J, L'Italien J J, Pilacinski W P, et al. High-level expression in Escherichia coli of biologically active bovine growth hormone [J]. DNA, 1985,4:273
收稿日期：1999-07-05，修回日期：1999-07-20, http://www.100md.com
单位：欧阳菁 杨林 杨洁 龙綮新 王珣章(中山大学生物医药中心、生物防治国家重点实验室，广州 510275)；魏平华 谢明权(广东省农业科学院，广州 510650)
关键词：猪生长激素基因；序列分析；杆状病毒载体系统；表达
中国病毒学000313摘要：通过对猪生长激素(pGH)基因的cDNA进行测序，得到pGH cDNA的全序列，并与Seeburg等报道的序列进行了比较和讨论。然后利用具人工合成启动子和多角体蛋白XIV启动子的转移载体质粒pSXIVVI+X3/4构建出含pGH基因的重组质粒pX3/4-pGH。将pX3/4-pGH与致死缺失型线性化AcMNPV-OCC- DNA共转染Sf9细胞，构建出既能形成多角体又能表达pGH基因的苜蓿丫纹夜蛾核多角体重组病毒AcMNPV-pX3/4-pGH-OCC+。感染重组毒株的Hi 5细胞可溶蛋白及其培养上清的SDS-PAGE和Western blot的分析结果表明，感染细胞的蛋白电泳带的20.7 kDa处有一条猪生长激素特异带，但培养上清中没有。凝胶黑度扫描估测结果显示pGH蛋白占细胞可溶蛋白的4.48%。
, 百拇医药
中图分类号：Q786， S859.793 文献标识码：A
文章编号：1003-5125(2000)03-0285-06
Expression of Porcine Growth Hormone Gene
in Baculovirus Vector System
OUYANG Jing，YANG Lin，YANG Jie，LONG Qing-xin，WANG Xun-zhang
(Biopharmaceutical Center, State Key Laboratory for Biological Control,Zhongshan University, Guangzhou 510275, China)
, 百拇医药
WEI Ping-hua，XIE Ming-quan
(Guangdong Academy of Agricultural Sciences, Guangzhou 510650, China)
Abstract: The cDNA sequence encoding porcine growth hormone (pGH) was inserted into the plasmid of pBluescript M13(-). The sequening results showed that a 984 base-pair fragment is identical to that reported by Seeburg et al., with the exception of one different base-pair in the ORF. Then, a baculovirus recombinant vector (pX3/4-pGH) with the pGH ORF was constructed and cotransfected Hi5 cells with the linearized parental virus (AcMNPV-OCC-) DNA to produce the recombinant virus AcMNPV-pX3/4-pGH-OCC+, in which the pGH gene was driven by the synthetic and XIV promoters. The SDS-PAGE and Western blot analysis showed that the pGH gene product, 20.7 kDa fusion protein, was obtained. The expressed pGH accumulated up to about 4.48% of the soluble cellular proteins as estimated by CS-930 scanning of the SDS-PAGE gel stainning by Coomassie Brilliant Blue R250.
, http://www.100md.com
Key words: Porcine growth hormone; Sequence analysis; Baculovirus vector system; Expression
基金项目：广东省自然科学基金重点项目(963007)
作者简介：欧阳菁(1975年-)，女，湖南省人，硕士研究生，研究方向为病毒基因的调控
参考文献
[1] 苏悌之，Oxender D L, R1-Geweley M E，等.猪生长激素cDNA重组及在大肠杆菌中表达[J].生物化学与生物物理学报，1990，22(2)：111-120
[2] 余旭平，齐顺章.在大肠杆菌中温度诱导高效表达猪生长激素[J].生物工程学报，1991，7(4)：307-311
, 百拇医药
[3] O'Reilly D R, Passarelli A L, Goldman I F， et al. Characterization of the DA26 gene in a hypervariable region of the Autographa califormica nuclear polyhedrosis virus genome [J]. Journal of General Virology, 1990,71:1029-1037
[4] O'Reily D R, Miller L K, Luckow V A. Baculovirus expression vetors, A laboratory Mannul [M]. New york: WH Freeman, 1994
[5] Maeda S, Kawai T, Obinata M, et al. Production of human α-inferferon in silkworm using a baculovirus vector [J]. Natrue, 1985,315:592-594
, 百拇医药
[6] 王NFDB4章，谢伟东，龙綮新，等.形成多角体的杆状病毒载体系统的建立[J].病毒学报，1991，7(3)：253-261
[7] Sambrook J, Pritsch E F, Maniatis T, Molecular Cloning: A Laboratory Manual [M]. 2nd Ed, Cold Spring Harbor: Cold Spring Harbor Press, 1989.
[8] Summers M D, Smith G E. A manual of methods for baculovirus vectors and insect cell culture rocedures, Texas Agricultural Experiment Station Bulletin [J]. 1985,(555):27-30
[9] Seeburg P H, Sias S, Adelman J, et al. Efficient bacterial expression of bovine and porcine growth hormone [J]. DNA, 1983,2:37-45
[10] George H J, L'Italien J J, Pilacinski W P, et al. High-level expression in Escherichia coli of biologically active bovine growth hormone [J]. DNA, 1985,4:273
收稿日期：1999-07-05，修回日期：1999-07-20, http://www.100md.com