抗乙肝病毒表面抗原PreS1(20-47)单链抗体基因克隆及其在大肠杆菌和烟草中的表达
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摘要
乙肝病毒表面抗原PreS1(20-47)表位是重要的乙肝病毒(HBV)表位，其识别肝细胞受体，并与HBV侵染人肝细胞有关。高亲和的抗乙肝病毒表面抗原PreS1(20-47)抗体具有封闭HBV的作用。噬菌体展示技术提供了获得结合特异表位抗体的新途径。我们用乙肝病毒表面抗原PreS1(20-47)体外免疫淋巴细胞，构建了抗PreS1(20-47)人源免疫抗体库。所获得的单链抗体的重链属人抗体V_H4亚族，轻链属人抗体V_λ4亚族，经检测，确定该单链抗体能与PreS1(20-47)特异结合，其亲合常数在10~(-7)-10~(-8)M范围内，具有潜在的应用价值。
用大肠杆菌和烟草分别表达编码抗乙肝病毒表面抗原PreS1(20-47)表位单链抗体。融合硫氧还蛋白的单链抗体在大肠杆菌BL21(DE3)中以包涵体的形式得到高效表达，单链抗体表达量达占30％的总蛋白以上。经变性复性，用固相金属离子亲和色谱法一步分离纯化了具有功能的单链抗体，在一升的发酵液中得到78mg的重组蛋白。
在用烟草作为生物反应器时，分别将该单链抗体靶向细胞质和内质网。经Western Blot分析，靶向细胞质中表达时，可溶单链抗体最高占总的可溶蛋白的0.06％。而靶向内质网中表达时，表达量达到总可溶蛋白的0.5％。以固相金属离子亲和色谱法，从转基因烟草中一步分离到纯度达90％的具有生物活性的单链抗体，每克叶片组织可获得9μg重组蛋白。
在水培条件下，转基因烟草根可连续分泌具有活性的重组抗乙肝病毒表面抗原PreS1(20-47)单链抗体进入到液体培养液中，不须破坏植物即可连续获得重组单链抗体，为利用植物生物反应器连续生产单链抗体开辟了新途径。
PreSl (20-47) is one of the important epitope of HbsAg, which has been pointed out to have relationship with infection of HBV. Humanized antibody against PreSl with high affinity is a new way to block HBV. In addition, the display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities. Here we explored the use of this technology to find human antibodies with biological properties. A phage-scFv specific for PreS 1 (20-47),
with an affinity of 1x10-7 -1x10-8M, was isolated from a phage display immune library, which was constructed with PBLs immunized by PreSl conjugated to BSA in vitro. Sequencing of the scFv showed that heavy chain belongs to VH4 and light chain belongs to V A 4 subgroup respectively. This study obtained the high affinity human scFv against PreS 1(20-21) for the first time. It is the foundation of therapy of Hepatitis B.
The gene of scFv was cloned into the expression vector pET32a(+) and subsequently expressed in Escherichia coll. The expressed scFv fused with the 109 aa Trx-Tag thioredoxin protein was above 30% total cell protein in the shaking flask cultures. After dissolving inclusion bodies, renaturing inclusion bodies and further purification by immobilized metal ion affinity chromatography (IMAC), the fusion protein Trx-scFv of electrophoretic purity was obtained, they still possessed antigen-binding activity.
Plants offer various advantages for the production of pharmaceutical proteins over conventional production systems such bacterial or mammalian cell culture. In order to explore transgenic plants for production of recombinant scFv specific for PreSl (20-47), Nicotiana tabacum was transformed with a gene encoding anti-PreSl of hepatitis B surface antigen scFv and bearing an //-terminal endoplasmic reticulum protein signal peptide sequence. High accumulation of the scFv protein in transgenic tobacco plants was achieved by retention of the recombinant antibodies in the lumen of the endoplasmic reticulum (ER). Expression levels of scFv antibodies reached up to 0.5% of total soluble proteins in leaves. The biologically active scFv was easily purified (to 90% purity) from SAFH4 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the SAFH4 plant line indicates that 9 (JL g of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.
The production of the scFv antibody proteins in plant root exudates was also addressed. The scFv antibody protein was continuously secreted from the transgenic tobacco roots into a simple hydroponic medium at 630 to 760 ng g-1 dry weight of root day-1. The antibody was about 2% of the total secreted protein and still possessed antigen-binding activity. It is a potential way to product recombinant scFv proteins.
用大肠杆菌和烟草分别表达编码抗乙肝病毒表面抗原PreS1(20-47)表位单链抗体。融合硫氧还蛋白的单链抗体在大肠杆菌BL21(DE3)中以包涵体的形式得到高效表达，单链抗体表达量达占30％的总蛋白以上。经变性复性，用固相金属离子亲和色谱法一步分离纯化了具有功能的单链抗体，在一升的发酵液中得到78mg的重组蛋白。
在用烟草作为生物反应器时，分别将该单链抗体靶向细胞质和内质网。经Western Blot分析，靶向细胞质中表达时，可溶单链抗体最高占总的可溶蛋白的0.06％。而靶向内质网中表达时，表达量达到总可溶蛋白的0.5％。以固相金属离子亲和色谱法，从转基因烟草中一步分离到纯度达90％的具有生物活性的单链抗体，每克叶片组织可获得9μg重组蛋白。
在水培条件下，转基因烟草根可连续分泌具有活性的重组抗乙肝病毒表面抗原PreS1(20-47)单链抗体进入到液体培养液中，不须破坏植物即可连续获得重组单链抗体，为利用植物生物反应器连续生产单链抗体开辟了新途径。
PreSl (20-47) is one of the important epitope of HbsAg, which has been pointed out to have relationship with infection of HBV. Humanized antibody against PreSl with high affinity is a new way to block HBV. In addition, the display of repertoires of antibody fragments on the surface of filamentous bacteriophages offers a new way of making antibodies with predefined binding specificities. Here we explored the use of this technology to find human antibodies with biological properties. A phage-scFv specific for PreS 1 (20-47),
with an affinity of 1x10-7 -1x10-8M, was isolated from a phage display immune library, which was constructed with PBLs immunized by PreSl conjugated to BSA in vitro. Sequencing of the scFv showed that heavy chain belongs to VH4 and light chain belongs to V A 4 subgroup respectively. This study obtained the high affinity human scFv against PreS 1(20-21) for the first time. It is the foundation of therapy of Hepatitis B.
The gene of scFv was cloned into the expression vector pET32a(+) and subsequently expressed in Escherichia coll. The expressed scFv fused with the 109 aa Trx-Tag thioredoxin protein was above 30% total cell protein in the shaking flask cultures. After dissolving inclusion bodies, renaturing inclusion bodies and further purification by immobilized metal ion affinity chromatography (IMAC), the fusion protein Trx-scFv of electrophoretic purity was obtained, they still possessed antigen-binding activity.
Plants offer various advantages for the production of pharmaceutical proteins over conventional production systems such bacterial or mammalian cell culture. In order to explore transgenic plants for production of recombinant scFv specific for PreSl (20-47), Nicotiana tabacum was transformed with a gene encoding anti-PreSl of hepatitis B surface antigen scFv and bearing an //-terminal endoplasmic reticulum protein signal peptide sequence. High accumulation of the scFv protein in transgenic tobacco plants was achieved by retention of the recombinant antibodies in the lumen of the endoplasmic reticulum (ER). Expression levels of scFv antibodies reached up to 0.5% of total soluble proteins in leaves. The biologically active scFv was easily purified (to 90% purity) from SAFH4 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the SAFH4 plant line indicates that 9 (JL g of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.
The production of the scFv antibody proteins in plant root exudates was also addressed. The scFv antibody protein was continuously secreted from the transgenic tobacco roots into a simple hydroponic medium at 630 to 760 ng g-1 dry weight of root day-1. The antibody was about 2% of the total secreted protein and still possessed antigen-binding activity. It is a potential way to product recombinant scFv proteins.
引文
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[9] Neurath AR, Kent SBH, Strick N, Parker K. Identirecation andchemical synthesis of a host cell receptor binding site on hepa-titis B virus[J]. Cell 1986, 46: 429-438.
[10] Adreone P, Carsaro C, Gramenzi A. Heptatology, 1996, 24: 774-777.
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