［ Med Sci］ 华 中 科 技 大 学 学 报 ［ 医 学 （ 英 德 文 ） 版 ］ Journal of Huazhong University of Science and Technology

26 (5): 497-499, 2006

497

Construction of Human ScFv Phage Display Library against Ovarian Tumor

夏劲松

毕 昊

姚 琴

屈 伸

宗义强

XIA Jinsong ( )1 , BI Hao ( )2 , YAO Qin ( )2 , QU Shen ( )2 # , ZONG Yiqiang ( )2 1 Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong Universty of Science and Technology, Wuhan 430030, China 2 Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

：

Summary In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E. coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 109 cfu/µg was obtained. After amplification with helper phage, the titer of antibody library reached 5 1012 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor. Key words ScFv phage display library; ovarian tumor; SOE; radioimmunoimaging DOI 10.1007/s 11596-006-0502-y

×

×

：

Radioimmunoimaging (RII) is a new radio-imaging technique, in which, specific binding of radionuclide with tumor is led by anti-tumor antibody, becoming the hot spot of diagnosis and research of tumors. The result of RII is related with antibody quality. Presently, most antibodies used in RII are murine hybridoma antibodies, which can produce the human antimouse antibody (HAMA) reaction[1] and some techniques such as hybridoma technique are very complicated and time consuming, hybridoma cell can t be conserved for long time and passage time is limited, so the application of RII in clinical diagnosis is hindered[2]. The birth of phage antibody library technique[3] brings a new strategy for the preparation of human monoclonal antibody, which doesn t need cell hybridization, so the defect of unstability of hybridoma cell line is overcome. The new method is simple and low cost and the most significant advantage of it is that antibody can be human resource, which overcomes HAMA reaction, so it can benefit the application of RII. In this study, the ScFv library against ovarian tumor was constructed successfully in the first time, which laid a foundation for the screening of ScFv against ovarian tumor for the RII diagnosis.

’

’

1 MATERIALS AND METHODS 1.1 Materials Plasmid pCANTAB5E, E. coli TG1 and helper phage M13KO7 were purchased from Pharmacia Co. XIA Jinsong, male, born in 1973, M.D., Ph.D. Corresponding author * This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30200295). #

(USA). TRIzol reagent and reversed transcriptive enzyme (M-mlv), dNTP, Oligo(dT) were purchased from Promega Co. (USA). TaqDNA polymerase, T4DNA ligase, restriction enzymes Sfi and Not , agarose gel DNA purification kit were purchased from TaKaRa (Japan). Lymphocyte isolation agent was purchased from Promega Co. (USA). PCR primers were synthesized by Cyber Co. (China). 1.2 Extraction of Total mRNA from Lymphocytes Total mRNA was extracted from lymphocytes in peripheral venous blood, lymphonod of ovarian cancer patients, lymphocytes in fresh cord blood according to the instruction of TRIzol reagent manual. 1.3 Amplification of VH and VL Gene of Antibody A set of nest PCR primers for VH and VL gene of antibody were used according to the referenus 4,5. cDNA was obtained by inverse transcription as template. Firstly, PCR was performed by outer primers with condition: 94 1 min, 48 2 min, 72 2 min, 25 cycles to obtain DNA fragment containing VH or VL gene respectively and used as templates for the next round PCR with inner primers for VH and VL gene. The condition for VH was 94 1 min, 61 1 min, 72 1.5 min, 35 cycles and for VL was 94 1 min, 55 1 min, 72 1.5 min 35 cycles. The resulting VH and VL DNA fragments were recovered by agarose gel DNA purification kit. 1.4 Construction of ScFv by SOE VH and VL DNA fragments were connected by SOE to obtain ScFv[4,5]. The reaction conditions were: same amount of VH and VL DNA fragments were mixed as template for the SOE. In the firstly 5 runs, primers were absent, 94 1 min, 55 1 min, 72 1.5 min, 5 cycles, then primers were added for next 30 cycles, 94 1 min, 61 1 min, 72 1.5 min.

Ⅰ

℃×

℃×

×

℃× ℃×

Ⅰ

℃×

℃× ，

℃×

℃× ℃×

℃× ℃×

℃× ℃×

℃×

℃

498

Journal of Huazhong University of Science and Technology

1.5 Construction of ScFv Library The resulting ScFv were recovered by agarose gel DNA purification kit and digested by Sfi and Not and inserted into plasmid pCANTAB5E. The recombinants were electrotransformed into competence TG1 and incubated for 1 h at 37 in swing bed, and then seeded on the SOBAG medium plate (SOB medium containing 100 mg/L ampicilin and 20 g/L glucose) at 30 for 24 h. The positive colonies were picked up randomly for the restriction enzyme analysis by Sfi and Not . The transformation efficiency was detected to calculate the capacity of ScFv phage antibody library. All positive colonies were washed in SOBAG medium and incubated at 37 for 1 h in swing bed (250 r/min). When log phase reached, helper phage M13KO7 were added and incubated for 1 h. By centrifugation, infected TG1 were collected and resuspended in 2 YTAK medium (2 YTA medium containing 100 mg/L ampicilin and 50 mg/L kanamycin) at 37 for 12 h in swing bed (250 r/min). By centrifugation, the supernatant was collected, which contained ScFv phage antibody library. 1.6 Detection of the Titer of ScFv Library The supernatant containing phage antibody was concentrated by PEG and diluted at 10－6, 10－8, 10－10 and 10 － 12 concentration gradient for bacteriophage plaque sum detection. The titer of ScFv phage antibody library was detected according to the number of bacteriophage plaque.

Ⅰ

℃

Ⅰ

Ⅰ

×

［Med Sci］ 26 (5): 2006

Ⅰ

3 109 cfu/µg. The restriction enzyme analysis by Sfi and Not showed that in most positive colonies, ScFv were inserted into vector pCANTAB5E exactly (fig. 4).

Ⅰ

℃ Ⅰ

℃

℃

×

×

Ⅱ;

Fig. 2 Amplified VL DNA M: DNA Marker Lane 1 by nest PCR

－4: Amplified V

L

DNA

2 RESULTS 2.1 Amplification of VH and VL Gene By the primers for different subgroups, the nest PCR successfully amplified the different VH and VL DNA fragments. VH was about 420 bp and VL about 320 bp respectively (fig. 1 and fig. 2).

Ⅰ

Ⅱ

Fig. 3 Electrophoresis of VH , VL and ScFv M: DNA Marker ; Lane 1: ScFv; Lane 2: VH; Lane 3: VL

－

Fig. 1 Amplified VH DNA M: DNA Marker ; Lane 1 4: Amplified VH DNA by nest PCR

2.2 Construction of ScFv by SOE VH and VL DNA fragments were successfully connected by SOE and the electrophoresis result showed that obtained ScFv was about 700 bp which was accorded with anticipated 740 bp[4, 5] (fig. 3). 2.3 Construction of Human ScFv Library According with the transformation efficiency, the capacity of ScFv phage antibody library was supposed to

Ⅱ

Ⅰ

Ⅰ Ⅳ

Fig. 4 Restriction enzyme analysis by Sfi and Not M1: DNA Marker ; M2: DNA Marker ; Lane 1: ScFv; Lane 2: positive colony digested by Sfi and Not Lane 3: vector pCANTAB5E digested by Sfi and Not

Ⅰ; Ⅰ

Ⅰ

Ⅰ

2.4 Detection of Titer of Human ScFv Library The titer of obtained human ScFv Library was 5 1012 cfu/mL according with titer detection.

×

Journal of Huazhong University of Science and Technology

［Med Sci］ 26 (5): 2006

3 DISCUSSION Human ScFv can be made by phage antibody library technique with many merits, such as small molecular weight, low immunogenicity, quick clearance, high penetrating ability, high T/NT ratio and low background in the developing, which has shown a strong superiority in the RII. The common procedure of the construction of human phage antibody library includes extraction of mRNA from human peripheral blood lymphocyte or lymphonode firstly, as the template for the amplification of VL and VH gene, then the C end of VL and N end of VH are connected to form ScFv gene. The VL and VH genes can be amplified from immunized lymphocytes and resulting antibody library has a high affinity but small capacity. Naive library obtained from un-immunized lymphocytes has big capacity and multiplicity but small specificity and affinity[6]. In this study, lymphocytes of ovarian cancer patients were used for the construction of antibody library in order to get antibody with high affinity. Meanwhile, lymphocytes of fresh cord blood were used as complementary materials to enrich the multiplicity of antibody library in order to get antibodies with different affinity for the research of the relationship between conformation and affinity by comparing their gene sequence. The primers design for VL and VH gene is crucial for the multiplicity of antibody library. Most primers were designed according to the Kabat immunoglobulin data bank, such as: 1. primers at specific sequence in FR1and FR4[5, 7, 9]; 2. primers at specific sequence in antibody gene family; 3. primers at 5 end of lead sequence and 3 end of J region. In our study, in order to get high amplification efficiency, nest PCR was used and outer primers were at 5 end of lead sequence and 3 end of J region and the inner primers were at FR1 and FR4[4]. The connection of VH and VL gene by SOE to form ScFv was the difficult step in the construction of antibody library. A good connecting result was attributed to the purity and equal amount between VH and VL. Linker used to construct ScFv had many types, in our study, the common linker, (Gly4Ser)3[4, 5], a peptide containing 15 amino acids was used. Transforming efficiency is a critical factor affecting the capacity of antibody library. In the construction of antibody library, electrotransformation was adopted. For the higher transforming efficiency, the conditions for electrotransformation including concentration of compe-

′

′

′

′

499

tence, amount of DNA and electric field intensity were optimized (data not shown). Finally, we obtained antibody library with capacity being 3 109 cfu/µg. The restriction enzyme analysis showed that most colonies contained ScFv. In our study, ScFv library against ovarian tumor was constructed successfully in the first time, which laid a foundation for the screening of ovarian tumor specific ScFv as a good protocol for the RII diagnosis or research for ovarian tumor.

×

REFERENCES 1 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. 1975. Journal of immunology, 2005,174:2353-2355 2 Shawler D L ,Bartholomew R M, Smith I M et al. Human immune response to multiple injections of murine monoclonal IgG. Journal of Immunology, 1985, 135:15301535 3 Barbas C F 3rd, Kang A S, Lerner R A et al. Assembly of combinatorial antibody libraries on phage surfaces: the gene site. Proceldings of the national Academy of Sciences of the Vnited States of America, 1991,88: 7978-7982 4 Jin He, Ge Zhou, Kang-Da Liu et al. Construction and preliminary Screening of a Human phage Single-chain Antibody Library Associated with Gastic Cancer. Joural of Surgical Research, 2002,102:150-155 5 Waterhouse P, Griffith A D, Johnson K S et al. Combinatorial infection and in vivo recombination: A strategy for making large phage antibody repertoires. Nucleic Acids Research, 1993,21:2265-2266 6 Marks J D, Hoogenboom H R, Bonnert T P et al. By-passing immunization: human antibodies from V-gene libraries displayed on phage. J Mol Biol, 1991,222(3):581-597 7 Larrick J W, Danielsson L, Brenner C A et al. Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction. Biochem Biophs Res Commun, 1989,160(3):1250-1256 8 Huston J S, Levinson D, Mudgett-Hunter M et al. Protein engineering of antibody binding sites: recovery of specific activity in anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA, 1998,85:5879-5883 9 Kabat EA, Wu TT, Perry H M et al. Sequence of proteins of immunology interest. US Dep Health Hum Serv, Public Health Serv Natl Inst Health, 1991,1229-1231 (Received Sep. 15, 2006)

Ⅲ