Expression，Purification and Bioactivity Detection of Recombinant Hum an IL-12 in Argyogramma agnata

xu Jin—ping; MENG Xiao-lin“; WANG Jian; LU Wei

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August 2002

xu Jin—ping, MENG Xiao-lin“, WANG Jian and LU Wei. Expression，Purification and Bioactivity Detection of Recombinant Hum an IL-12 in Argyogramma agnata[J]. Virologica Sinica, 2002, 17(4): 340-343.

Citation: xu Jin—ping, MENG Xiao-lin“, WANG Jian, LU Wei. Expression，Purification and Bioactivity Detection of Recombinant Hum an IL-12 in Argyogramma agnata .VIROLOGICA SINICA, 2002, 17(4) : 340-343.

重组人白介素一12在银纹夜蛾中的表达纯化及其生物活性

1.
武汉大学病毒研究所，湖北武汉430072

摘要

KB细胞经PDBu刺激，采用异硫氰酸胍一步法提取细胞总RNA。RT—PCR法获得重组人白介素一12(rhlL一12) P35和P40 cDNA。将hlL一12 P35 cDNA和P40 cDNA分别克隆到pAcUW51载体的Polyhedrin和P10启动子下游，构建pAcUW51一IL12转移载体。pAcUW51一ILl2与坏死缺陷型线性苜蓿银纹夜蛾核型多角体病毒基因DNA 共转染Sf9细胞，获得重组杆状病毒Ac—hiLl2。该病毒经血腔感染银纹夜蛾幼虫．采用亲和层析法纯化rhlL一12； SDsPAGE(银染法)、Western blot鉴定表达和纯化的产物；ELISA检测rhlL一12含量；MTT法检测rhlL一12样品生物活性。rhlL。12分子量为75kD。rhlL一12在Sf9细胞培养中表达水平为17．8 g／10 细胞；在银纹夜蛾幼虫中表达水平为200—300mg／L血淋巴。纯化的重组rhlL-12样品对经PHA—P激活的PBMC有明显的增殖活性．且具有明显的促NK细胞杀伤活性的生物活性。
- 人白介素一12
- , 表达
- , 纯化
- , 生物活性
- , 银纹夜蛾

Expression，Purification and Bioactivity Detection of Recombinant Hum an IL-12 in Argyogramma agnata

1.
Institute of Virology．Wuhan University。Wuhan 430072。China

Abstract

Two cDNA fragments encoding human interleukin一12(hII一12)P35 and P40 subunits were isolated by RT．PCR from KB cells stimulated with PDBu，and then cloned into pCR ．1 respcetively and down the double promoters of pAcUw 5 1．The recombinant baculovirus Ac—hiL2 was obtained by cotransfecting with pAeUW 5 1一ILl2 and baeuloGold M Linearized baculovirus DNA．Argyrogramma agnata larvae were infected with recombinant baculovirus Ac—hiLl2 by hemoeoel injection．The rhII， 1 2 was purified from the supernatant with affinity chromatography．The blood lymph supernatant har— vested and rhII，12 purified were SUbjected to SDS-PAGE(silver stain)and Western blot．The level of rhIL-12 was detected by EU SA．Bioactivity of purified rhII，12 samples were detected by M 1vr method．It is indicated that Ae-hlL12 can replicate in fat body and midgut cells of Argyrogramma Agnata larvae．The MW of rhlL一12 expressed was 75kD．The expresion levels of rhIL-12 were 17．8 ／~g／lO cells and 200—300mg／L in Sf9 cells and Argyrogramma agnata Staudinger larvae blod lymph respectively．The purified rhII，12 has significant activities which enhanced NK eytotoxieity and increased the proliferation of human PBMC PHA-P activated significantly．
- Human interleukin-12
- , Expression
- , Purification
- , Bioactivity
- , Argyogramma agnata Staudinger

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Expression，Purification and Bioactivity Detection of Recombinant Hum an IL-12 in Argyogramma agnata

1. Institute of Virology．Wuhan University。Wuhan 430072。China

Abstract: Two cDNA fragments encoding human interleukin一12(hII一12)P35 and P40 subunits were isolated by RT．PCR from KB cells stimulated with PDBu，and then cloned into pCR ．1 respcetively and down the double promoters of pAcUw 5 1．The recombinant baculovirus Ac—hiL2 was obtained by cotransfecting with pAeUW 5 1一ILl2 and baeuloGold M Linearized baculovirus DNA．Argyrogramma agnata larvae were infected with recombinant baculovirus Ac—hiLl2 by hemoeoel injection．The rhII， 1 2 was purified from the supernatant with affinity chromatography．The blood lymph supernatant har— vested and rhII，12 purified were SUbjected to SDS-PAGE(silver stain)and Western blot．The level of rhIL-12 was detected by EU SA．Bioactivity of purified rhII，12 samples were detected by M 1vr method．It is indicated that Ae-hlL12 can replicate in fat body and midgut cells of Argyrogramma Agnata larvae．The MW of rhlL一12 expressed was 75kD．The expresion levels of rhIL-12 were 17．8 ／~g／lO cells and 200—300mg／L in Sf9 cells and Argyrogramma agnata Staudinger larvae blod lymph respectively．The purified rhII，12 has significant activities which enhanced NK eytotoxieity and increased the proliferation of human PBMC PHA-P activated significantly．