Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

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CHEM. RES. CHINESE UNIVERSITIES 2008, 24(6), 767-770 Article ID 1005-9040(2008)-06-767-04

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Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody ZHANG Hai-hong, ZHANG Xi-zhen, ZHAO Dong-hai, SHI He-liang, W Yong-hui, WU Yong-ge, W Xiang-hui* and KONG Wei* Vaccine Research Centel; College ofLife Science, Jilin University, Changchun 130012, P R. China Abstract Survivin, a novel member of inhibitor of apoptosis(1AP) protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass(A4J of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni2+affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained. Keywords Clone; Survivin; Expression; Polyclonal antibody

1 Introduction Survivin"], the smallest member of the inhibitor of apoptosis protein(1AP) family, is highly expressed in virtually all types of cancers, as well as in vascular endothelial cells during tumor associated angiogenesis but is undetectable in nonproliferating normal adult tissues"]. The cancer of overexpression of survivin shows its stronger aggressive behavior, such as the decreased response to chemotherapeutic agents compared with cancers that are survivin negati~e'~', which suggests survivin has a potential role in tumorigenes i ~ ' ~and ' , it has become a promising target for vaccination p ~ r p o s e s ' ~ , ~ ' . The researches about survivin are under intense state in cancer gene therapy. However, why this protein is upregulated and what is the molecular mechanisms of regulation in cancer remain largely unknownL71. Cloning and expression of human survivin and preparation of its polyclonal antibody are helpful to further researches for the molecular mechanism of survivin to inhibit apoptosis and understand the significance for clinical practice. In this study, human survivin cDNA was cloned

into the prokaryotic expression vector pRSET-B, and the recombinant expression plasmid, pRSET-Bsurvivin(pRSET-B-Surv), was constructed. Recombinant protein was expressed and purified, and survivin polyclonal antibody with high titer was obtained.

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Experimental

2.1 Materials 293 cells, E. coti strains TOP10 and BL21, and pRSET-B vector were obtained from our laboratory. The pGEM-T easy vector was purchased from Promega. TRIzol reagent was purchased from Lifetechnologies. Restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were purchased from New England Biolabs. Freund's complete adjuvant(FCA) and Freund's incomplete adjuvant(FL4) were purchased from Dingguo. The mAb against survivin was purchased from Novus-Biologicals. DL2000 marker and 1 kb DNA marker were purchased from Takara. Prestained protein marker was purchased from BioLabs and unstained protein molecular weight marker was purchased from Fermentas.

*Corresponding author. E-mail: [email protected]; [email protected] Received November 7, 2007; accepted January 8, 2008. Copyright 0 2008, Jilin University. Published by Elsevier Limited. All rights reserved.

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2.2 Methods 2.2.1 RNA Isolation and Reverse TranscriptasePolymerase Chain Reaction(RT-PCR) Total RNA was isolated from 293 cells by the method of Horikoshi and Sakakibara[*]. Total RNA was extracted from 293 cells, and cDNA of survivin was synthesized by reverse transcription using SuperScriptTMreverse transcriptase kit(Gibco). First-strand synthesis was performed using random hexanucleotides as primer, and PCR was performed with for-

ward(5'-CTgCAgTCgACgCCgCCACCATgggTgCCCCgACgTTg-3') and reverse(5'-ggATCCggTACCAAgCTTAATCCATggCAgCCAgC-3') primers at annealing temperature adjusted to 55 "C. PCR products were subsequently examined by agarose gel electrophoresis, yielding a 465 bp product. 2.2.2 Plasmids Construction The cDNA of survivin amplified from total RNA of 293 cells by RT-PCR were cloned into the pGEM-T vector. After confirming the size and sequence of survivin cDNA by restriction enzyme digestion and sequencing the recombinant plasmid pGEM-T-Surv, the cDNA of survivin was subsequently subcloned into PstIlHindIII sites of pRSET-B vector. 2.2.3 Expression, PuriJication, and IdentlJication of Suwivin The recombinant express plasmid pRSET-B-Surv was transformed into E. coli BL21. The recombinant protein with N-terminal with 6His-tagged survivin was induced by 1 mmoVL isopropyl-/?-D-thiogalactopyranoside(1PTG) at an optical density of 0.4-0.6 at 37 "C for 5 h's expression. Bacterial lysates were prepared by sonication in buffer A(50 mmol/L Tris-HC1, pH 7.8, and 300 mmol/L NaCI, 8 mol/L urea). After centrifugation at 12000 r/min for 30 min, the supernatant was applied to a Ni2'-nitrilotriacctate (Ni-NIA) agarose column(QIAGEN), washed with buffers B and C(their composition is the same with buffer A but with pH 6.0 and 5.3, respectively). Then, the recombinant protein was eluted with buffer D(its composition is the same with that of buffer A but with pH 4.0). The eluted solution was detected by SDS-PAGE, and Western blot with the mAb against survivin(NovusBiologica1s). 2.2.4 Western Blot Analysis Survivin recombinant protein was degenerated by loading buffer( 100 mmoVL DTT, 0.1% Bromocresol

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Blue, 2% SDS, 50 mmol/L Tris-HC1 pH 6.8, 10% glycerol) and boiled for 5 min and then centrifuged for 10 min. The protein was loaded onto the gel and submitted to SDS-PAGE, and the resolved proteins were transferred to nitrocellulose membrane. After blocking with 3% nonfat milk in PBS for 40 min, the membrane was slit into equal width and incubated with various primary antibody for 90 min at room temperature. The blot was then probed with AP-conjugated secondary antibody for 40 min and stained by NBT and BCIP. 2.2.5 Preparation and IdentiJication of Antisurvivin PoEyclonal Antibody Polyclonal antibody against human survivin was generated via a standard immunization protocol whereby rabbits were immunized with purified recombinant survivin protein. Briefly, the purified fusion protein was used to immunize rabbits with 300 pg once a week by subcutaneous multiple sites injection on backside. The serum was collected from the marginal of ear vein before prime immunization as negative control; the serum was collected once a week after the fifth immunization. The rabbits were immunized seven times in all, and antiserum was collected three times. One week after the last injection, the rabbits were sacrificed and the serum was collected. The specificity and titer of all the serum samples were analyzed by Western blot in different dilution.

3 Results 3.1 Amplification of Human Survivin cDNA Fragment

The human survivin cDNA fragment was amplified by RT-PCR. The RT-PCR products were analyzed by means of electrophoresis with 1% Agarose gel. The fragment is 465 bp, and the size of the PCR product as shown in Fig. 1 is the same as that expected.

Fig.1 Agarose gel electrophoresis analysis of RT-PCR product of survivin cDNA Lane I: survivin cDNA produced by PCR lane 2: DL2000 marker.

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vector has been successfully constructed. 3.2 Construction of Expression Vector pRSETB-SUW

Plasmids pGEM-T-Surv and pRSET-B-Surv were both identified by restriction enzymes PstIIHindII. The results of agarose gel electrophoresis(Fig.2) and DNA sequencing demonstrate that the expression

Fig.2

Restriction analysis of recombinant vectors pGEM-T-Surv and pRSET-B-Surv

(A) Agarose gel electrophoresis of plasmid pGEM-T-Surv digested by PstIand Hindlll; (B) agarose gel electrophoresis of plasmid pRSET-B-Sun digested by Psfl and Hind 111. Lane 1: I kb DNA marker; lane 2: recombinant plasmid pRSET-B-Surv; lane 3: recombinant plasmid digested by PstI and Hind Ill.

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3.3 Expression, Purification, and Identification of Recombinant Survivin Protein

Blank vector pRSET-B and target vector pRSET-B-Surv were transformed into host E. coli BL21 and then induced by IPTG for 5 h. The target protein expressed by pRSET-B-Surv is a kind of fusion protein consisting of 6His and survivin. The relative molecular weight of the hsion protein is about 21 000[Fig.3(A)]. Western blotting analysis indicates a strong band at 21000 from the induced E. coli BL21 containing pRSET-B-Surv, and there is no band shown at the corresponding place in Western blotting analysis with antisurvin mAb as a primary antibody for the E. coli BL21 containing blank vector pRSET-B [Fig.3(B)]. The purified protein was analyzed by mean of SDS-PAGE[Fig.3(C)]. The results show that the recombinant protein was expressed and purified successfully.

SDS-PAGE and Western blot identification of recombinant protein

(A) SDS-PAGE stained by coomassie brilliant blue. Lane I : pRSET-B induced by IPTG for 5 h as negative control: lane 2: pRSET-B-Sorv induced by IPTG for 5 h; lane 3. prestained molecular weight marker: ( 8 ) Western blotting analysis with antisurvivin mAb as a primary antibody. Lane 1: prestained protein niolecular weight marker; lane 2: PRSET-B induced by IPTG for 5 h as negative controc; lane 3: PRSET-B-Surv induced by IPTG for 5 h; (C) purification of recombinant protein. Lane I : purified survirin protein; lane 2 : unstained protein molecular weight marker.

3.4 Preparation and Detection of Antisurvivin Polyclonal Antibody

A specific protein band was detected in the analysis of the antisera collected for the last three times by Western blot[Fig.4(A)] using the purchased antisurvivin mAb as positive control, which suggested that the rabbit immunized recombinant protein proFig.4 Western blotting analysis of specification and titer duced survivin antibody. of rabbit antisurvivin sera with purified recombiIn this article, we also investigated the titer of the nant survivin protein as antigen (A) Western blotting analysis for the specification of the fifth to the last antisurvivin antibody in the last antiserum[~ig.4(~)], boosts rabbit serum. Lane 1: negative control(serum of the rabbit before shows that the titer Of the immunization); lane 2: positive control(antisurvivin mAb); lanes 3-5: rabbit sera from the fifth to the last boosts; (B) Western blotting analysis for last antiserum was higher than that of the purchased the titer of the last immunized rabbit serum diluted as 1:1000(lane 2), Idb’ we the last time antiserum 1:5000(lane 3), and 1:50000(lane 4), and lane 1 is negative control(l:1000), in 1:1000, 15000, and 1:50000, respectively, then per lane 5 is positive control( I :ZOOO).

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formed Western blotting analysis. The result of the dilution 1:50000[Fig.4(B) lane 41 was still positive, which shows a high titer antisurvivin polyclonal antibody was obtained.

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Discussion

The human survivin gene produces a 16500 protein of 142 amino acids, and comprises three introns and four exons. Survivin is a recently discovered member of the IAPS~~'. It is composed of a single Baculovirus IAP Repeat(B1R) domain, and it does not contain a RING-finger domain that is found in other IAPs. It is unique among the IAP proteins and exhibits control of cell division and inhibition of apoptosis. One of the most significant features of survivin is its differential expression in cancer versus normal tissues. It is continuously present at excess levels in the majority of human tumor lung, breast, colon, gastric, oesophageal, pancreatic, liver, bladder, uterine, and ovarian cancers, suppressing apoptosis and regulating cell division. Previous have shown that survivin is associated with chemotherapy resistance, increases tumor recurrence, and shortens patient survival. S~ppression"~] of survivin function resulted in spontaneous apoptosis, enhancement of drug induced cell death, and inhibition of tumor growth in vitro and in vivo. These characteristics make it a useful tool in cancer prognos ~ s " ~ , 'and ~ ] ,therapy"8"91. The cDNA corresponding to full-length human survivin was amplified and subcloned into the His fusion protein expression vector pRSET-B, which offers N-terminal polyhistidine(6His) tag for the rapid purification of 6His fusion protein with Nichromatography. The relative molecule mass(MJ of expressed 6His-survivn fusion protein was 2 1000. The polyclonal antibody was prepared by the immunization of rabbits purified fusion protein. The Western

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blotting analysis shows that the last antiserum diluted to 1:50000 was still positive. These results reveals that a prokaryotic expression plasmid, pRSET-B-Surv, has been successfully constructed and a high titer and specificity antisurvivin polyclonal antibody has been obtained. Survivin has been a potentially promising genetic target for cancer immunotherapy. The molecular mechanism of survivin to inhibit apoptosis and the ef€icacy of antisurvivin vaccine therapy is not clearly understood. The successful expression of human survivin and preparation of its polyclonal antibody gave a basis for further research on the biological function and the clinical significance in cancer immunotherapy. References Johnson M. E., Howerth E. W., Vet. Pathol., 2004,41,599 Li F., Br . I Cancer, 2005,92,212 Myung A. L., Park G., Lee H. J., et al., BMC Cancer, 2005,5, 127 Nicola M. C., Nat. Rev. Cancer, 2004, 4, 837 Otto K., Andersen M. H., Eggert A,, et a/., Vaccine, 2005,23, 884 Wobser M., Keikavoussi P.,Kunzmann V., et al., Cancer Immunol. Immunother, 2006,55, 1294

Sah N. K., Khan Z., Khan G. J., et al., Cancer Lett., 2006,244(2), 64 Barbara V., Paul V., Simone W., et al., Hum. Pafhol.,2006,37,78 Do0 M. Sohn., Sung Y. K., Moo J. B., et al., Biomed. Pharmacother, 2006, 60(6), 289 Takai N., Miyazaki T., Nishida M., et al., Int. J. Mol. Med., 2002, 10,

211 Nadia Z., Marzia P., Maria G. D., J. Cell. Mol. Med., 2005, 9, 360 Seiji F., Louis M., Mol. Cancer Ther., 2006, 5, 1087 Haochuan L., Jerry Y ,Niederkoma S., et al., Ekp. Eye Res., 2006,83, 176 Akhtar M., Gallagher L., Rohan S., Adv Anat. Pathol., 2006, 13, 122 Parker A. S., Kosari F., Lohse C. M., et al., Cancer, 2006,107,37 Marioni G., Ottaviano G., Marchese R. R., Acta Otolaryngol., 2006, 126, 197 Abd El-Hameed A,, J. Egypt Natl. Canc. Inst., 2005,17,42 Lladser A,. Parraga M., Quevedo L., et al., Immunol., 2006,211, 11 Russo A,, Terrasi M., Agnese V, et al., Ann. Oncol., 2006, 17((Suppl.). 115