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丙型肝炎病毒非结构蛋白3人源单链可变区抗体的筛选与鉴定

来源:免疫学杂志 作者:风清扬 2009-2-21
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摘要: 丙型肝炎病毒非结构蛋白3人源单链可变区抗体的筛选与鉴定 免疫学杂志 2000年第4期第16卷 基础免疫学 作者:成军钟彦伟刘妍董菁杨继珍张玲霞 单位:成军(解放军302医院传染病研究所基因治疗研究中心,北京 100039)。噬菌体抗体 [摘 要] 目的 筛选、鉴定抗丙型肝炎病毒(HCV)非结构蛋白NS3的人单链可变区......


丙型肝炎病毒非结构蛋白3人源单链可变区抗体的筛选与鉴定

免疫学杂志 2000年第4期第16卷 基础免疫学

作者:成军 钟彦伟 刘妍 董菁 杨继珍 张玲霞

单位:成军(解放军302医院传染病研究所基因治疗研究中心,北京 100039);钟彦伟(解放军302医院传染病研究所基因治疗研究中心,北京 100039);刘妍(解放军302医院传染病研究所基因治疗研究中心,北京 100039);董菁(解放军302医院传染病研究所基因治疗研究中心,北京 100039);杨继珍(解放军302医院传染病研究所基因治疗研究中心,北京 100039);张玲霞(解放军302医院传染病研究所基因治疗研究中心,北京 100039)

关键词:噬菌体展示技术;丙型肝炎病毒;亲和筛选;噬菌体抗体

  [摘 要] 目的 筛选、鉴定抗丙型肝炎病毒(HCV)非结构蛋白NS3的人单链可变区抗体(ScFv),以解决人体内应用鼠单抗时的免疫原性问题,为进行抗HCV的基因治疗研究开辟新途径。方法 采用噬菌体表面展示技术,以重组的HCV非结构蛋白NS3为固相抗原,从噬菌体单链可变区抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性较强的HCV NS3 人单链可变区抗体的阳性克隆,并对其进行免疫检测及序列测定。结果 筛选出来的ScFv片段具有抗NS3的特异性。证实利用噬菌体抗体库技术,可以成功地获得HCV NS3人单链抗体ScFv的编码基因。结论 筛选获得了HCV非结构蛋白NS3的特异性单链抗体的编码基因。

  [中图分类号] R335[文献标识码] A

Screening and identification of a humanized single chain variable region antibody for hepatitis C virus non-structural 3 protein

CHENG Jun, ZHONG Yan-wei ,LIU Yan, DONG Jing, YANG Ji-zhen, ZHANG Ling-xia

  (Gene Therapy Research Center, Institute of Infectious Diseases, the 302 Hospital of PLA, Beijing 100039,China)

  [Abstract]Objective To screen and identify humanized single chain variable region antibody for hepatitis C virus non-structural 3 protein. Methods Phage display technique was employed in the screening and identification of humanized single chain variable region(ScFv) antibody by using recombinant hepatitis C virus (HCV) non-structural 3 protein as coating antigen. Results After 5 rounds of biopanning of a ScFv antibody phage library, 66 phage clones were evaluated by enzyme-linked immunosorbent assay(ELISA).2 phage clones were selected from 66 clones according to the highest OD value in the ELISA identification and lowest cross-reaction to the bovine serum albumin(BSA).The coding fragments of 2 ScFv were sequenced. After 5 rounds of biopanning of the ScFv phage library with initial 2.0×1013 clones, the HCV NS3 antigen-binding phage particles have been concentrated by 300 times. 66 phage clones were selected randomly for the subsequent ELISA screening and identification by using HCV NS3 and BSA as coating antigens, respectively. 2 phage clones were selected for their high A value in the ELISA identification and low cross-reaction to BSA. The 2 inserts in the phage vector were sequenced and confirmed its ScFv characterization. Conclusion We have successfully screened and identified 2 clones of HCV NS3 specific ScFv.

  [Key words] phage display; hepatitis C virus; biopanning; single chain antibody

  [Article ID]1000-8861(2000)04-0246-04

  Hepatitis C (HC) caused by hepatitis C virus(HCV) affects Chinese people frequently[1]. HCV infection not only can cause acute and chronic he-patitis, but has close relationship with the development of hepatic cirrhosis and hepatocellular carcinoma (HCC)[2]. Although interferon α (IFN-α) was the only world-wide recognized effective drug for the treatment for HC, many patients still were non-responsible to IFN-α treatment promptly in the clinical practice[3]. So it is a long term goal to pursue more effective treatment for HC and related diseases. The progress in molecular biology of HCV indicated that NS3 protein of HCV is a multiple functional protein with serine protease and RNA helicase activities. So the NS3 protein plays an important role in the HCV protein maturation, RNA replication and also has a potential role in the cleavage and activation of hepatocyte proto-oncogene, so HCV NS3 protein has close relationship with the development of HCC[4]. In order to de-velop efficient NS3 protease inhibitor, we apply the phage display technique using HCV NS3 protein as the immobilized antigen and succeeded in cloning specific ScFv antibody to HCV NS3 antigen.

  1 MATERIALS AND METHODS

  1.1 Materials Humanized ScFv antibody phage library in which the variable region coding genes of VL and VH were amplified by polymerase chain reaction(PCR) with degenerate primers and connected with a glycine linker[(Gly4Ser)3] was widely used in the screen and identification of humanized ScFv to various antigens[5]. The recombinant HCV NS3 protein was expressed and purified from engineered E.coli with routine methods. Purity of the recombinant HCV NS3 protein is more than 95% as demonstrated by SDS-PAGE. Phage M13K07 and plasmid DNA preparation kit were purchased from Pharmacia Co., Sweeden. Other reagents used in this experiment are all domestic products of analytical grade.

  1.2 Biopanning of a phage library The host E.coli TG1 was infected with phage M13K07, incubated at 37 °C for 12 hours, the phages in the supernatant were harvested and concentrated. Culture plate (Nunc) was coated with recombinant HCV NS3 protein at the concentration of 80 μg/ml. The coating buffer is 0.05 mol/L NaHCO3, pH9.6. The plate was blocked with BSA at the concentration of 10 g/L for 2 hours, and the concentrated phages were added to the wells of the plate, incubated at room temperature for 90 minutes. The plates were washed for 20 times with PBST and PBS buffer, respectively. The bound phages were eluted by the 0.1 mol/L of triethylamine, and neutralized with 1 mol/L Tris buffer (pH7.4). Recovered phages were used to infect the host E.coli TG1 at the log phase growth and HCV NS3 protein-binding phages were amplified. This biopanning, absorption-unbound-amplification proc-edure was repeated for 5 cycles.

  1.3 Identification of the phage clones After 5 rounds of biopanning, 66 phage clones were selected randomly. The clones were grown in 2 X TY medium with 100 mg/L of ampicilin at 37 °C overnight. The culture was continued at 30 °C overnight after adding helper phage. The supernatants were determined by ELISA for at least two times. The cross-reaction of the supernatants to the BSA antigen was conducted side by side. According to the ELISA results to the HCV NS3 and BSA antigen, 2 clones with high reaction to HCV NS3 and low reaction to BSA were selected.

  The plasmid DNA was prepared by using Wizard Plus midipreps DNA Purification System (Promega Co., USA) and sequenced with the primers of M13 (forward) and Lac Z(reverse) using ABI automated DNA sequencing machine (PE Co., USA).

  2 RESULTS

  2.1 Screening and enrichment of the phage li-brary Using HCV NS3 protein as the immobilized antigen, the humanized ScFv phage library was biopanned. In the 5 rounds of biopanning, the number of the binding phage particles to coated plates increased gradually. Finally, the phages binding to the coated plates were concentrated for about 300 times as indicated in table 1.

  2.2 Identification and characterization of selec-ted phage clones After the final round of biopanning, 66 phage clones were selected randomly and the secretion of the solvable ScFv antibody was confirmed by specific ELISA. The cross-reaction of these clones to BSA was also screened by the similar ELISA procedure. Among the 66 phage clones, 19 showed good reaction to the recombinant HCV NS3 protein with high OD value in the ELISA. In the cross-reaction screen, 6 among the 19 showed low cross-reaction with BSA(see figure 1). The combined results indicated that 2( a and c )of the 6 showed the highest reaction to HCV NS3 protein and lowest reaction to BSA. One of the 2 clones has been selected for further DNA sequence analysis.

Tab 1 Selection and enrichment of phagemid for each round of biopanning

selection

  round

No. of phagemid offered and recovered

  frequency of reactive/total phagemid

1 2.0×1013 3.0×108 1.5×10-5
2 2.0×1013 2.4×109 1.2×10-4
3 2.5×1012 2.6×109 1.1×10-3
4 3.0×1012 7.0×109 2.3×10-3
5 2.0×1012 9.0×109 4.5×10-3

Fig 1A values of 6 typical clones determined by ELISA

  2.3 Sequence analysis of two phage clones coding for the ScFv The DNA sequence analysis results indicated that the 2 clones of the humanized ScFv were identical to the reported ScFv results in the structural frames(see figure 2).

  3 DISCUSSION

  Phage display technique is a breakthrough tool in the study of the antibody after the hybridoma emerged[5]. Construction and screening of a phage library is a very efficient way to get humanized

Fig 2ScFv DNA sequence against HCV NS3 protein

  ScFv coding DNA sequence, and make it possible to express recombinant ScFv antibody by recombinant DNA engineering. Humanized ScFv techniques have many advantages[6]. This is the only method to get specific antibody bypassing the immunization step. The phage library screening not only makes the specific antibody selection come true, but also mimics the maturation procedure of the human antibody in vivo. So it is possible to get high affinity antibody from this selection. The ScFv selected from these library has a low molecular weight, can make it as a potential application in the real clinical diagnosis and treatment for both infectious diseases and cancer. Finally, phage display is an easy going method, which could be conducted in many laboratories. Until now, many ScFv were reported to various antigen by using phage display techniques. In this paper, we have got humanized ScFv antibody phage clones with high affinity to HCV NS3 protein, and low cross-reaction to BSA, which is commonly used in the protection of recombinant proteins. From 5 rounds of biopanning, the HCV NS3 specific phage particles were enriched for about 300 times. This biopanning procedure also mimics the maturation procedure for antibody in vivo. The primary results indicated that the HCV NS3 could be expressed by E.coli efficiently and recognized antigen in formalin-embedded tissue sections of the liver biopsy from the patients suffering from hepatitis C.

  Humanized HCV ScFv antibody has much potential use in the diagnosis and treatment for patients with hepatitis C.HCV NS3 protein is among the most important proteases of the structural and non-structural proteins of the HCV. As the protease inhibitors are playing an important roles in the treatment and control of human immunodeficiency virus (HIV) infection[7], HCV NS3 has also been regarded as an important target for anti-HCV drug design and selection[8]. In fact, mini-antibody is a hopeful reagent for the treatment of viral infections[9]. To use ScFv coding gene in the construction of a retroviral vector, also realized the inhibi-tion of HIV replication intracellularly in the HIV study. This method for the treatment of virus infection has been called ‘intracellular immun-ization’[10]. The recombinant adenoviral and retroviral vectors construction is under way in this laboratory. But it will be difficult to evaluate the efficacy of these vectors, for there is no good cell lines and animal models at present which could be used in the efficacy study for the HCV infections. But along with the development of the HCV infection cell and animal model development, the efficacy of the mini-body and intracellular immunization stra-tegy will play a very important part in the control of the HCV infection.

  [Foundation item]This work is supported by National Natural Science Foundation of China(39900130).

  [Biography]CHENG Jun(1964-),male,born in Gaoqing county,Shandong,associate professor,M.D.,field of research:gene cloning,gene therapy and gene vauine which is related to infections disease.

  [REFERENCES]

  [1] ZHANG JY, DAI M, WANG X, et al. A case-control study of hepatitis B and C virus infection as risk factors for hepatocellular carcinoma in Henan, China[J]. Int J Epidemiol, 1998,27(4):574-578.

  [2] PETRI TU, NOBUKO K, ZHOU D M, et al. Selection of RNA aptamers that bind specifically to the NS3 protease of hepatitis C virus[J]. Eur J Biochem, 1997,248(1):130-138.

  [3] KUMAR PKR, MACHIDA K, URVIL PT, et al. Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA[J]. Virology, 1997,237(2):270-282.

  [4] LAMARRE A, TALBOT PJ. Characterization of phage-displayed recombinant idiotypic antibody fragments against coronavirus neutralizing monoclonal antibodies [J]. Viral Immunol, 1997,10(1):175-182.

  [5] MARKS JD, HOOGENBOOM HR, BONNERT TP,et al. By-passing immunization, human anti-bodies from V-gene libraries displayed on phage[J]. J Mol Biol, 1991,222(3):581-597.

  [6] MORIYA K, FUJIE H, SHINTANI Y, et al. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice[J]. Nat Med, 1998,4(9):1 065-1 067.

  [7] HOOGENBOOM HR, BRUE AP, HUFTON SE, et al. Antibody phage display technology and its application[J]. Immunotechnol, 1998,4(1):1-20.

  [8] RONDON IJ, MARASCO WA. Intracellular anti-bodies (intrabodies) for gene therapy of infectious diseases[J]. Annu Rev Microbiol, 1997,51:257-283.

  [9] DIMASI N, MARTIN F, VOLPARI C, et al. Characterization of engineered hepatitis C virus NS3 protease inhibitor affinity selected from human pancreatic secretary trypsin inhibitor and minibody repertoires[J]. J Virol, 1997,71(10):7 461-7 469.

  [10] MARTIN F, VOLPARI C, STEINKUHLER C, et al Affinity selection of a camelized V(H) domain antibody inhibitor of hepatitis C virus NS3 protease [J]. Protein Eng, 1997,10(5):607-614.

[Received date] 1999-11-16; [Revised date]2000-01-04



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