抗SARS冠狀病毒S1蛋白N端249至667的單克隆抗體的制備與鑒定

1、抗sars冠狀病毒s1蛋白n端249至667的單克隆抗體的制備與鑒定作者：溫坤梅亞波丘立文i廖志勇袁國(guó)勇2；車(chē)小燕1c第一軍醫(yī)大學(xué)珠江醫(yī)院屮心實(shí)驗(yàn)室，廣東 廣州510282；彳香港大學(xué)微生物學(xué)系，香港）摘要：ei的 在獲得了具有免疫原性的sars冠狀病毒s1蛋門(mén)片段的基礎(chǔ) 上，制備和鞭定特異性抗該段s1蛋白單克隆抗體（mab） 0方法原核表達(dá) 含s蛋門(mén)受體結(jié)合區(qū)的sars冠狀病毒s1蛋門(mén)片段slc（n端249-667氨基 酸殘基），其免疫原性經(jīng)sars病人恢復(fù)期血清鑒定后免疫balb/c小鼠，按 常規(guī)方法制備社克隆抗體，并采jij elisa間接法、免疫熒光和免疫印跡進(jìn) 行篩選和鑒定。結(jié)果 篩

2、選出3株特異性針對(duì)saks冠狀病毒s1蛋門(mén)n端 249-667的mab雜交瘤細(xì)胞株，igg亞類(lèi)鑒定1株為iggl, 2株為igg2a, 經(jīng)免疫熒光鑒定與人冠狀病毒株229e和0c43無(wú)交叉反應(yīng)。結(jié)論 獲得3 株抗sars冠狀病毒s蛋白受體結(jié)合區(qū)特異性單克隆抗體，為建立新的sars 冠狀病毒檢測(cè)方法的和進(jìn)一步研究s蛋白的功能奠定了基礎(chǔ)。關(guān)鍵詞：sars冠狀病毒；單克隆抗體；刺突蛋白；受體結(jié)合區(qū)preparation and charactcrization of monoclonal antibodies againstsi domain at n-tcrminal residues 249 t

3、o 667 of sars-associatodcoronavirus si proteinwen kun1； met ya-bo1； qtu li-wen1； liao zhi-yong1； yuen kwok-yung2；che xiao-yan1"central laboratory, zhujiang hospital, first m訂itary medicaluniversity, guangzhou 510282, china; "department of microbiology,university of hong kong, hong kong, ch

4、ina.abstract: objective to prepare and characterize monoclonal antibodies (mabs) against si protein of severe acute respirato-ry syndrome (sars)-associated coronavirus (sars-cov).met hods 6-his-tagged recombina nt fmgme nt at n-terminal residues 249 to 667 of sars-cov si protein including s-protein

5、receptor-binding domain was expressed in e. coli. the im-munogenicity of this si domain was identified and used to immunize balb/c mice for the production of hybridomas. the identification of the mabs against this si domain was performed using indirect enzyme-1inked immunosorbent assay (elisa), indi

6、rect immunofluorescence assay (tfa) and western blotting, respectively. resui ts three hybridomas producing mabs spc-cific to the si domain was obtained, with a relative molecular mass of 48 500. none of the 3 mabs were reactive with human coronaviruses 229e and 0c43. two of the mabs were lgg2a isot

7、ype, and the other was iggl- conclusion this is the first re-port of mabs produced against s-protein recep to r-b in ding domain of sars-cov. the 3 sl-specific mabs may be useful for further study of the function of the s protein and for diagnosis of sars-cov infection.key words: severe acute respir

8、atory syndrome-associated coronavirus;sars-cov; monoclonal antibody; spike glycoprotoin;receptorebinding domainreceived: 2004-01-03this work is supported as the key medical research project for sarsprevention sponsored by the ministry of science and technology ofchina and by guangdong provincecorres

9、ponding author: che xiao-yan, m. d., professor in the centrallaboratory of zhujiang hospital, tel: 86-20-61643592, fax: 86-20-61643592, e-mai1: 1inchepub. guangzhou. gd. cn, chcxiaoyanyahoo. comthc two authors, wen kun and met ya-bo, have made equal contribu-tions to this worka novel coronavirus has

10、 been identified as the ma-jor cause of sever acute respiratory syndrome (sars)123, but the biological func tions of thi s sars-associated cor on av i rus (sars-cov) remain currently poorly char-acterized recent studies reported that angiotensin-con-verting enzyme 2 (aec2) could be a functional rece

11、ptor for sars-cov and the receptor-binding domain (rbd) was identified in the n-terminal residues 17 to 274 of the si spike protein of the virus 4. subsequent studies demonstrated that the rbd was locatod in tho n-tormi-nal residues 303 to 5375 or residues 318 to 510 6 the si protein is also found t

12、o be a target for inducing neu-tralizing antibody responses to sars-cov infection7. in spite of the fact that the function of s protein is not fully understood, si protein seems to play a key role in the initial virus infection. based on this understanding, the monoclorml antiodies(mabs) targe ted a

13、t si domain can be a potentially useful tool for the st udy of the function of s protein and for the diagnosis of sars. fur-thermore, the availability of blocking antibodies may be meaningful in the treatment of sars.in this paper, we report the cloning, expression and antigenic characterization of

14、the various fragments of si domain as well as the preparation and characteriza-tion of mabs against the recombinant protein sic de-rived from the si protei n con tai ning then-terminal residues 249 to 667.materials and methodsvirus and cell lineshuman coronavirus strains 229e (no. vr740), 0c43 (no.

15、vr759) and cell lines vero e6 (no. crl-1586), mrc-5 (no. ccl-171), bs-c-1 (no. ccl-26) were all purchased from american type culture collection (atcc).plasmidsthe plasmid encoding si protein gene, pet-28b (+)/si (40-2 001 bp, hku-39849), was kindly provid-ed by the department of microbiology, univer

16、sity of ilong kong. the expression vector pqe-30 and m15 strain of escherichia coli were purchased from qia-gen.reagentsthe restrict!on endonucleases bamht and hindttt were purchased from new england biolabs, and kpnt, pstt, extaq, dntp and dna ladder were from takara. ni resin was obtained from qia

17、gen. freund" s adju-vant, 50% peg 1450 solution, hat and iit were pur-chased from sigma aldrich. goat anti-mouse igm, igg, iggl, lgg2a, lgg2b and lgg3 peroxidase conjugates, goat anti-human igg peroxidase conjugates, goat an-ti-mouse tgg fttc conjugatc and ami noethyl carbazole (aec) single sol

18、ution were the products of zymed. rpmi 1640 culture medium and fetal bovine serum were from gibco-brl. all the chemical reagents used in this study were of analytical grade, and prepared with ion-depleted water.sera and animalsserum samples of sars patients for this study were serologically confirme

19、d by inclirect immunofluo-rescence and enzyme-linkeel immunosorbent assay (elisa)8, and the sera from healthy donors were used as control for the same assay. five 6-week-old female balb/c mice were provided by the experimental ani-mal center of first mil itary medical univcrsity.cloning si fragments

20、 into the expression vector and its expressionsi gene encoding the amino acid residues 14 to 667 of the spike protein of the sars-cov was ampli-fied by pcr using the following primers: 5'-cgggatc cagtgaccttgaccggtgcaccac-3' 5'-cgggg taccttaacgtaataaaga/actg-3, . the si gene was digested

21、with bamlll and kpnl and liga ted into the corresp on ding res tricti on sites in pqe-30 plasmid to prepare the pqe-30/s1. three gene fragments respec-tively encoding the amino acid residues 14 to 24& 249 to 445 and 249-667 of the si spike protein derived from si protein were obtained by digesti

22、on of the recombi-nant plasmid (pqe-30/s1) with the restriction endonu-cleases, all ligated into the pqe-30 plasmid according to their restriction sites in the frame emd upstream of the series of 6 histidine residues and designated as pqe- 30/sla, pqe-30/slb and pqe-30/slc respectively. the plasmids

23、 were transformed into competent m15 cells following the protocol of qiagen manual. the strai ns con tain ing the recomb inemt plasmids were cultured in luria-bertani medium (lb medium) with continuous shaking at 37 °c till the 00 value of the medium reached 0. 7, foil owed by induetion with lm

24、mol/l isopropyl- p -d-thiogalactopyranoside (iptg) at 30 °c for 4 h.purification and western blot analysis of si fragmentsthe cultured cells were centrifugecl and harvested. 6-his-tagged recomb i nant fragments were purified us-ing ni res in un der den atur ing con di tion accord ing to the man

25、u fac tu rer，s instructions and analyzed by wes tern blot ting. the purified proteins were separated by sodi-um dodecyl sulfate (sds)-po1yacry1amide gel electro- phoresis (page), and electrophoretically transferred to polyvinylidene fluoride (pvdf) membranes with the method described previously9. af

26、ter the transfer, the membranes were blocked in pbs-t containing 7% de-fattecl milk for 2 h at room temperature and then incu-bated respectively with the sera from sars patients and healthy donors at the dilution of 1 ： 100 in the blocking buffer for 12 h at 4 °c. after 6 washes, the membranes

27、were incubated with peroxidase-conjugated goat an-ti-mouse igg at the dilution of 1 : 500 for 1 h at room temperature. the membranes were incubated with aec signal solution for 10 min at room temperature after suf-ficient washing and the react i ons term in ated with clis-tilled water.tmmunizationst

28、he specific immunogcnicity of the fragments des-ignatod sic was characterized by wes tern blotting using the sera from sars patients and healthy donors respec-tively. the immunization was performed as described previously10. briefly, 5 female 6-week-old balb/c mice were immunized in the rear footpad

29、s and subcuta-neons tissues with purified recombinant sic (50 u g pro-tein per mouse) emulsified with an equal volume of complete freund's adjuvant for the first injection and with incomplete freund" s adjuvant for the following 3 booster injections. the sera sampled from the eyepit of the

30、mice after the fourth imm un iati on were tested by elt sa to i de ntify the mouse with the strongest response to sic. three days before fusion experiment, the identi-fied mouse was injected with 100 u g sic protein intra-venously without the adjuvant.production and screening of hybriclomasthe 96-we

31、ll culture plates were prepared for the fusion by adding 5x 10 normal mouse splenocytos in rpmi 1640 medium supplcmented with hypoxanthine- aminopterin-thvmidine medium into each we11 on the day before the fusion. the plates were incubated in 5% co2 at 37 °c, and hybridomas were induced by fusi

32、ng 1x10“ immunized mouse splenocytes and 2x10* myelo-ma cells (nst ; atcc) with 50% peg 1450 solution. the culture supernatants of the hybridoma cel is were screened for anti body produc tion by indirect elisa with recombina nt protein and lysates of the mutant strain of sars-cov as the coating anti

33、gens, respective-ly. positive hybridoma clones were selected and cloned by limiting di lution till the positive rate reached 100%. the cell lines were then frozen and stored in liquid ni-trogen.determination of the immunoglobulin subclassthe microtiter plates were coated with purified sic protein (1

34、00 ng/well) for 12 h and blocked with 3% bovine serum albumin. the cell culture supernatant was added into each well in dupl icate (100 u 1/well) and in-cubatod at 37 °c for 1 h, followed by the addition of goat anti-mouse igg, igm, iggl, igg2a, igg2b and igg3 peroxidase conjugates (diluted at

35、1 ： 1 000, 100 u 1/well) and further incubation at 37 °c for 30 min. te-tramethylbenzidine was used as the substrate. the reac-ti on was term in ated with 0 5 mol /l h2so1 and the ab-sorbance measured at 450-nm wavel ength in a mi-croplate elisa reader.western blot analysis of mabsthe experimen

36、t was carried out following the same procedure as described above. purified recombinant sic protein was separated on sds-po1yacry1ami de gels un-der reduced condition and the gel was transferred to pvdf membranes the membranes were incubated with the mab from hybridoma culture supernatants. af-ter b

37、eing washed with pbs containing 0. 5% tween-20, the membranes were incubated with goat anti-mouse tgg peroxidase conjugate. aec chromogen was used for signal detection.if analysis of the mabsvero e6 cells infected with sars-cov, mrc-5 cells infected with human coronavirus 229e, bs-ct cells infected

38、with human coronavirus 0c43 were har-vested and washed twice in cold pbs. the cells were deposited on 8-10 well chamber slides, air-dried and fixed in 30% methanol and 70% acetone at -20 °c for 10 min. after being washed with pbs, the slides were incubated with hybridoma culture supernatants at

39、 37 °c for 1 h and further incubated with goat anti-mouse fitc conjugate at 37 °c for 30 min. the slides were stained with 0. 25% evans blue/pbs buffer and examined with a leica ecl ipse cpifluorescent microscope.resultsexpression and purification of si fragments and antigenic characteriza

40、tion by western blot analysisexpression of 6-histagged recombinant fragments derived from si protein were observed in e. col i m15 treated with tptg. expression of complete si protcin was not soon in e. coli. the fragments containing amino acid residues 14 to 248, 249 to 445 and 249 to 667 of the si

41、 protein, were successfully expressed and purified (fig. 1). among the three fragments, designated as sla, sib and sic respectively, only the fragment sic reacted with the sera of 12 sars patients in westorn blotting. a prominont immunorcactivc protoin band of sic (with relative molecular mass of 48

42、 500) was seen in the western blot ting (fig. 2), whereas the sla and sib frag-me nts failed to exhibit reaction with the sera of the sars patients (data not shown). these results suggest that the antigenic domain is located between n-tcrminal amino acid residues 249 to 667 of the si spike protein.w

43、jxlo1) 12397.466.243.031.02023 wr(xlo*)97.466.243.031.0bfig.1 sds-page analysis of the 3 purified si fragments panel a lane 1: a fragment containing amino acid residues 14 to 248 of si protein, designated as sla; lane 2:low-mo1ecu1ar-mass protein marker; lane 3: a fragment containing amino acid resi

44、dues 249 to 445 of si protein, designated as sib; panel b lanes 1, 2: a fragment containing amino acid residues 249-667 of si protein， designated as sic;lane 3: low-mo1ocular-mass protein markerproduction of mabs to si proteinhybridomas producing antibodies that are specifi-cally reactive with the r

45、esidues 249 to 667 of si protein were successfully established from splenocytes of the mice immunized with purified recombinant sic protein. fusion of the splenocytes of immunized mice with nst myeloma cells produced more than 100 hybridoma cell lines, of which 3 (a50, b30 and c31) were selected on

46、the basis of their strong reactivity with the sic protein as shown by elisa and if. the isotypes of the mabs a50, b30 and c31 were iggl, igg2a and igg2a, re-spectively.wjxlq j 22201 997 '664530mj143fig. 2 western blot analysis of the 3 purified si fragments purified sic protein (10 u g/gel) was

47、separated on 10% polyacrylamide gel and electroblotted onto pvdf membrancs. the strips were incubated with either convalescent sars patients' sera or control sera; panel a: the band about 48 500 in relative molecular mass shows that sic protein is specifically recognized by the sera from 12 sars

48、-cov infection patients; panel b: sic protein does not react with the sera from 12 health subjects.characterization of the mabs to si protein by western blotting and ifas shown in fig. 3, the mabs specifically bound to recombinant sic protein. it can be noted that the mabs produced a single band wit

49、h the sic protein at exactly the same location (about 48 500 in relative molecular mass) as the reaction band of the serum from convalcs-ccnt sars patient. strong reaction of the antibodics with sars-cov-inf ectcd vero e6 cells was observed in indirect immunofluorescence assay, without their recogni

50、tion of human coronaviruses 0c43, 229e (fig. 4), ccv or 1bv (data not shown). these results suggest that the mabs were specific to sars-cov with no cross-reactivity with other coronaviruses.34 5678 m.(xlo')9766453020.1fig.3 western blot analysis of the mabs againt purified recombinant, sic prote

51、inpurified sic protein was separated on a 10% polyacrylamide gel and elec troblot ted onto a pvdf membrane. the st rips were incubated with different anti-sic protein monoclonal antibodies from hybridoma cell culture supernatants. lanes 1-3: different mab clones (a50, b30, c31); lanes 4, 5: anti-his

52、tidine antibody and mouse anti-slc polyclonal serum; lanes 6: serum of a convalescent sars patient; lanes 7, 8:negative controls (anti-human igm and anti-sars-covrecombinant n protein mabs from hybridoma cell culturesuper rmtants)dtscusstonthe s protein is bel ieved to play a crucial role in viral e

53、ntry into the target collsll. recent studies have shown that the si domain of sars-cov is character-ized as the binding molecule on thesurface of the virus particles4 5 6. in our study, we found that antigenic do-main of si protein is located between n-terminal amino acid residues 249-667, including

54、 the s-protoin reccptor- binding domain at residues 318-510. the identifiedtion of the functional si domain has allowed us to carry out further study of this virus.fig.4immunofluorescence of mab c31 tosars-cov-infected vero e6 cells (a, x400), human-cov 229e-infected mrc-5 cells (b, x200) and 0c43-i

55、nfected bs-c-1 cells (c, x200), with normal vero e6 cells (d, x200) used as the negative controlthe cells were stained with hybridoma cell culture superrmtants followed by goat anti-mouse tgg fttc conjugate.the results were examined with epifluorescent microscope, showing speci f ic comb in at i on

56、of the mabs wi th sars-cov without cross-reaction with human-cov.in this study, we have produced and characterized 3 mabs specific for si domain at residues 249-667. the isotypes of the 3 mabs were identificd as tggl, tgg2a and igg2a respectively, and all of them recog-nized si domain with a relativ

57、e molecular mass of 48 500. indirect immunof1uorescence assay showed that the mabs reacted* strongly to sars-cov-infectcd vero e6 cells, without cross-reaction with human corona- viruses 0c43 and 229e.so far to our knowledge this is the first report of mabs against s-protein receptor-binding domain

58、of sars-cov produced by using purified recombinant si protein as the immunogen. the development of mabs against s protein using the lysato of sars-cov as the immunogen was not successful in our previous studies, suggesting t hat the crude virus is very likely the im-munogen rat her t han the predomi

59、nant antigen of the virus, given that the n protein is the major vi ral an tige n produced in cor on avirus - infected cells12, and therefore the majority of the inabs (about 80%) should be target-cd at the n protein in our previous studies. thus, the re-combinant protein as the immunoge n may facil it ate the development of sensitive and specific mabs.in our previous studies, we establi