| Overexpression of HER2(Human epidermal growth factor receptor2) has beenassociated with tumorigenesis of breast cancers which is the second common cancerin women considering the morbidity and the mortality. About25%of the breastcancer patients with high level HER2expression have poor prognosis and lesssensitivity to endocrine therapy. The tumors expressing high level of HER2seemmore likely to relapse and metastasize. So it is important to detect the level of HER2in order to guide clinical treatment.Currently, there are two drugs are in clinical treatment for the breast cancer. Oneis Herceptin, a humanized monoclonal antibody capable of binding extracellulardomain of HER2. Another is Lapatinib, a small molecule capable of inhibitingintracellular tyrosine kinase. Both drugs to some extent can prolong the survival of thepatients and improve the response rate to chemotheraphy.To guide the clinical treatment, HER2specific antibodies are used to detectHER2expressed in breast cancer cells. Routinely, the antibodies are prepared frommouse or rabbit.IgY is avian immunoglobulin equivalent to mammalian IgG and can begenerated in egg yolk as yolk antibody. Yolk antibody exists in the yolk of egg as thematernal antibody during the early life of avain. Isolating antibodies from avians’ eggsis the most common way to abtain IgY. Within all avians, chichen is widely used toprepare IgY. IgY isolated from chicken has the following advantages:1) unable tobind mamalian Fc receptor；2） inert to activate mamalian complement system；3）shows low affinity to Staphylococcus aureus protein A and Streptococcal protein G.Therefore, when used diagnostically, IgY can effectively reduce the background of thereaction system and avoid of false positive result. Besides, the Fc domain of chickenIgY can be stably conjugated to biotin, enzymes or fluorescent materials. Theevolution distance between avian and mamalian makes chicken a good host inproducing antibodies against mamalian proteins. IgY is isolated from eggs that can be harvested daily. Comparing to generate IgY, preparation of antibody from mammaliananimals has the disadvantages such as repeated bleeding and animal sacrifice. ThusIgY can be used to replace the mammalian antibody, and be a better alternative inpractical usage. Forthermore, IgY is advantageous to be abtained in a big amoutcheaperly.Based on the above-mentioned, we prepared the polyclonal yolk antibodiestargeting the extracellular domain of HER2(HER2-ECD). The purpose of us is toprepare yolk antibodies recognizing HER2and to establish a platform for yolkantibody preparation.Several steps used to prepare yolk antibodies against HER2-ECD are as follows.1、 Expression and purification of recombinant HER2-ECD protein（rHER2-ECD） in E.coliHER2-ECD gene was abtained from the pcDNA3-GFP-HER2-ECD, a plasmidcarrying a fusion gene between green flurencent protein (GFP) and human epidermalgrowth factor receptor2extracelular domain (HER2-ECD), and subcloned intopET-28a. To purify, the C-terminal of HER2-ECD was constructed with His-tag(6×Histidine). The resultant plasmid, pET-28a-HER2-ECD, was transformed into theBL21(DE3), a Ecoli. cell for expressing recommbinat HER2-ECD (rHER2-ECD).After being induced with IPTG, the Ecoli.were harvested and ultra-sonicated. Theresultant lysate was detected by SDS-PAGE, showing that one third of rHER2-ECDwas expressed in inclusion body and the rest was in soluble form. The rHER2-ECD inthe lysate was purified with nickel affinity chromatography. Briefly, the harvestedE.coli was lysed in the lysis buffer containing8mol/L ure and then ultra-sonicated.After centrifugation, the supernatant of the lysate was loaded to the nickel affinitycolumn. The elute was subsequently added to the G-25gel filtration column toremove the salts. The eluted protein in the first peak was analysed by SDS-PAGE,revealing a70KD band and a30KD band. The protein in the70KD band wasrecognized by anti-His-tag antibodies in Western blot, indicating the protein in theband was rHER2-ECD with His-tag.2、Preparation and comparation of specific yolk antibodies and serum antibodiesagainst rHER2-ECDrHER2-ECD was used to immunize the hens to produce HER2-ECD specific IgY(IgY-HER2). To determine the dosage of rHER2-ECD for immunization, we noticed that1μg/injection of bovine serum albumin (BSA) usged to immunize hens toproduce high level of IgY. So, rHER2-ECD at2μg/injection plus adjuvant206wasused to immunize hens on day0，14and21. After the first shot, the sera and eggswere collected weekly to compare the concentration of HER2-ECD specific IgY inthem. PEG precipitation was used to isolate yolk antibody as described below. Brifely,1）20ml PBS was mixed with10ml yolk and then votexed. PEG was added into themixture at the final concentration of12%;2）After being votexed, the mixture wascentrifuged and the supernatants were collected and added with PEG at the finalconcentration of3.5%. Votexed and centrifuged again. Discarded the supernatant;3）Precipitants were dissolved in10ml PBS, and PEG was added to reach theconcentration of3.5%，the resultant solution was centrifuged and the supernatant wasdiscarded;4) repeated step3, the final pellets containing IgY was dissoved in2mlPBS.Indirect ELISA was used to detect the IgY in the serum and yolk, showing thatthe level of HER2-ECD specific IgY in the yolk is similar as that in the serum on day30after the first immunization. Before day30, the level of serum HER2-ECD specificIgY was higher than those in the egg. Afterwards, it reversed. On day42after the firstimmunization, the titers of IgY from yolks of one immunized hens reached to1:1280.The same level of HER2-ECD specific IgY in another hen was achieved on day49after the first immunization.The70KD band and the30KD band from the SDS-PAGE were both recognizedby yolk antibodies in Western blot, thus revealing the rHER2-ECD specific yolkantibody (IgY-HER2) arising.3、To detect whether the IgY-HER2could recognize the HER2positive breastcancer cellsThe lysates of two HER2positive cells (ZR-75-30and BT474) were preparedseparately and both were recognized by IgY-HER2in Western blot, showing theIgY-HER2could recognize the HER2expressed in the cells of tumor, thus illustratingyolk antibodies against rHER2-ECD are suited for detection of HER2and HER2positive tumor cells.rHER2-ECD also was expressed in the baculovirus expression system (BEVS).BEVS has been using to express various of recombinant eukaryotic proteins with modifications nessassary for the functions of the proteins. The BEVS was chosen toexpress rHER2-ECD.The HER2-ECD gene was obtained from the pcDNA3-GFP-HER2-ECDmentioned above and subcloned into the transfer vector pFastBacTM1to construct thepFastBacTM1-HER2-ECD plasmid. The resultant pFastBacTM1-HER2-ECD wastransformed into the DH10Bac, a Ecoli. cell carrying a Bacmid with a DNA fragmentderived from the genome of the baculovirus AcMNPV genome for homologousrecombination. The DH10Bac with the Bacmid carrying the HER2-ECD gene werescreened by blue-white selection and verified with PCR. The selected clone(DH10Bac-Bacmid-HER2-ECD) was used to express rHER2-ECD.In sum, rHER2-ECD was successfully expressed in E.coli and used to immunizehens for raising rHER2-ECD specific IgY both from yolk and serum. The IgY couldrecognize cancer cells overexpressing HER2, indicating the yolk antibodies can beused to detect HER2positive tumor cells and are promising alternatives of themammalian counterpart antibodies for detecting HER2positive breast cancer, with theadvantages of easy preparation and causing less damage to annimals. In theexperiments, BVES was later used for expressing rHER2-ECD with a aim ofabtaining rHER2-ECD with the nessessary modifications such as glycosylation. |
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