应用随机多肽文库分析BAC5单抗相关的抗原表位

目的　鉴定能被BAC5单抗识别的定位于鼻咽癌细胞表面的抗原表位。方法　应用BAC5单抗作为靶抗体对噬菌体呈现的随机 12肽文库进行 3轮生物淘洗 ,用抗体捕获和竞争试验的夹心ELISA方法选择和鉴定阳性噬菌体克隆 ,对阳性和阴性噬菌体的外源性DNA片段进行序列分析 ,推导和比较由这些噬菌体所呈现的多肽氨基酸序列。结果　通过 3轮生物淘洗能被抗体捕获的噬菌体克隆为 77% ( 35 /45 )。用竞争试验从所捕获的克隆中测得 8个阳性克隆。来自这 8个克隆的噬菌体呈现三种外源多肽 ,即$CH Q S H Y P Y P V V S L ( 4/8)$CQ N Q A W F S Q P P V R M ( 3/8)和 T Q A Y K G F P V L P S ( 1/8) ,与来自阴性克隆的多肽序列 N H Q S T F W Q K W T A ( 6 /6 )比较 ,前 3个序列在靠近肽的N端都具有相同的脯氨酸 (P)和缬氨酸 (V)结构 ( P V )。结论　BAC5单抗能识别近N端含有脯氨酸和缬氨酸结构的多肽。这些多肽可能模拟存在于鼻咽癌细胞表面并与BAC5单抗相关的抗原表位的构象。     

随机十二肽噬菌体展示文库筛选血型B抗原模拟多肽的实验研究

目的:筛选出替代血型B抗原的模拟多肽,用多肽抗原替代糖类抗原.方法:抗血型B抗原的单克隆抗体作为固相筛选靶分子,对随机十二肽噬菌体展示文库进行生物淘选(bio-panning),经包被-结合-洗脱-扩增等循环3轮,对筛选的克隆ELISA鉴定,并通过剂量依赖实验验证其结合特异性.最后提取DNA测序,确定模拟肽氨基酸序列.结果:3轮筛选结束,得到2个亲和力较强的十二肽序列TKNMLSL-PVGPG和HSLKHTQMSYSS.结论:经过生物筛选得到模拟多肽序列,利用噬菌体展示技术筛选糖类抗原的模拟肽具有可行性,为糖类抗原的研究提供一种新思路.     

胃癌细胞特异性结合肽的筛选及鉴定

目的 筛选与胃癌细胞特异性结合的多肽。方法 以正常细胞为吸附细胞,胃癌细胞为筛选靶细胞对噬菌体随机12肽库进行消减筛,用细胞酶联免疫吸附试验（ELISA）、免疫细胞和组织化学法及裸鼠正常组织结合实验鉴定阳性克隆并进行DNA测序。结果 经三轮筛选,利用ELISA从随机挑选的24个噬菌体克隆中得到8个与胃癌细胞具有高结合力的噬菌体阳性克隆,经免疫细胞化学及裸鼠正常组织结合试验鉴定,发现第20、24两个克隆能与胃癌细胞特异性结合,而不与正常细胞和裸鼠组织结合,噬菌体阳性克隆氨基酸序列无同源性。结论 得到两个序列不同的特异性结合胃癌细胞的噬菌体克隆,这可为进一步的研究提供实验依据。     

胃癌腹膜高转移细胞特异性结合噬菌体多肽的筛选及鉴定

目的寻找能够与胃癌腹膜高转移细胞GC9811-P特异性结合的噬菌体多肽,探索治疗胃癌腹膜转移的新方法。方法运用噬菌体呈现肽技术,先后用胃癌的腹膜高转移细胞系GC9811-P和其亲本细胞GC9811对噬菌体12肽库进行消减性的全细胞淘洗．经过3轮筛选,随机挑选40个噬菌体单克隆C1～G40。用ELISA法选取能够与GC9811-P特异性结合的单克隆。将选出的单克隆分别注入裸鼠腹腔,采用免疫组化法排除与正常组织亦高结合的阳性单克隆。对筛选出的噬菌体克隆进行DNA序列测定,并推导其外源性氨基酸序列,进行同源性分析。结果经过3轮淘洗,噬菌体克隆得到理想富集。C9、C18、C23、C29、C34和C37可与GC9811-P特异性结合,经免疫组化证实,这6个单克隆均不与裸鼠腹腔内正常组织结合。测序结果大致展示了两种外源性多肽,即TLNINRLIIPRT和SMSIxSPYIxxx。结论筛选出6个可与GC9811-P细胞特异性结合的噬菌体多肽;这两个肽序列能否阻断GC9811-P细胞向腹膜转移尚待进一步确定。     

噬菌体随机12肽库筛选肝癌患者血清肿瘤标记物的初步研究

目的:从噬菌体随机多肽文库中,筛选出能与肝癌患者血清特异性结合的短肽分子.方法:采用肝癌患者血清作为配基,筛选以融合蛋白形式在丝状噬菌体M13外壳蛋白Ⅲ表达的随机12肽文库.按吸附一洗脱一扩增的淘筛过程,经3轮淘筛后,随机挑取噬菌体克隆用ELISA检测其特异性,评价分析其诊断肝癌的价值.结果:经3轮淘筛后,特异性结合的噬菌体富集增加近100倍.用.ELISA检测第3轮筛选后随机挑取的单个噬菌体克隆,其中特异性最好的3个克隆具有诊断肝癌的潜在价值.结论:噬菌体展示肽库技术,可以有效进行肝癌相关抗原肽的筛选研究,为获得特异性诊断试剂进而为肝癌的诊断提供依据.     

噬菌体随机12肽库筛选肝癌患者血清肿瘤标记物的初步研究

目的：从噬菌体随机多肽文库中,筛选出能与肝癌患者血清特异性结合的短肽分子.方法：采用肝癌患者血清作为配基,筛选以融合蛋白形式在丝状噬菌体M13外壳蛋白Ⅲ表达的随机12肽文库.按吸附一洗脱一扩增的淘筛过程,经3轮淘筛后,随机挑取噬菌体克隆用ELISA检测其特异性,评价分析其诊断肝癌的价值.结果：经3轮淘筛后,特异性结合的噬菌体富集增加近100倍.用.ELISA检测第3轮筛选后随机挑取的单个噬菌体克隆,其中特异性最好的3个克隆具有诊断肝癌的潜在价值.结论：噬菌体展示肽库技术,可以有效进行肝癌相关抗原肽的筛选研究,为获得特异性诊断试剂进而为肝癌的诊断提供依据.     

噬菌体肽库在宫颈癌血清肿瘤标志物筛选中的应用

目的:用噬菌体随机12肽库对宫颈癌患者血清进行差异性筛选,筛选出能与宫颈癌患者血清特异性结合的肿瘤标志物。方法:应用噬菌体随机12肽库对宫颈癌患者和正常人血清进行三轮差异性筛选,用ELISA法检测噬菌体克隆对宫颈癌患者血清结合的特异性。结果:经过三轮筛选,噬菌体富集率逐轮提高,提高近100倍。用ELISA法对50例宫颈癌患者血清和50例健康者血清检测验证,其中获得一株与宫颈癌患者血清特异结合较好的噬菌体,对宫颈癌的早期诊断具有潜在价值。结论:筛选出能与早期宫颈癌患者血清高亲和力特异结合的短肽,为研究宫颈癌肿瘤标志物及宫颈癌的早期诊断奠定了基础。     

MDA-MB-231乳腺癌细胞特异性多肽的初步筛选

目的 利用噬菌体展示技术,从噬菌体随机十二肽库中筛选出能够特异性结合MDA-MB-231乳腺癌细胞的噬菌体克隆.方法 以人正常乳腺细胞为减性筛选细胞、MDA-MB-231乳腺癌细胞为靶细胞,对噬菌体随机十二肽库进行筛选,挑取富集后的阳性单克隆噬菌体,酶联免疫吸附试验（ELISA）及DAB染色鉴定阳性噬菌体的特异性及亲和力.结果 经过3轮筛选,噬菌体得到约113倍的富集,随机挑选11株单克隆噬菌体,ELISA显示8号噬菌体单克隆对乳腺癌细胞的亲和力是对照的6.5倍,DAB鉴定亦显示其对乳腺癌细胞的特异性及亲和力最高,命名为LK-8.结论 利用噬菌体筛选技术成功筛选出能够特异性结合MDA-MB-231乳腺癌细胞的特异性噬菌体单克隆LK-8,可为进一步合成特异性多肽用于早期诊断和靶向治疗乳腺癌奠定基础.     

人表皮生长因子受体2模拟表位的筛选

目的:从噬菌体12肽库中筛选出人表皮生长因子受体2（Her2）的抗原模拟表位。方法:以曲妥珠单抗为靶分子,在噬菌体12肽库中进行3轮淘选,以ELISA方法及竞争抑制实验鉴定阳性克隆,并对阳性克隆株进行测序。结果:经过3轮淘选,与曲妥珠单抗结合的噬菌体得到了有效富集,回收率从（2.00×10-8）%增加到（2.87×10-5）%,ELISA显示20个克隆中筛选获得了18个与曲妥珠单抗具有较高亲和性的阳性噬菌体,对阳性克隆测序获得两种氨基酸序列:HTSSLWHLFRST、VHWDFRQWWQPS。结论:噬菌体展示技术可成功筛选到表皮生长因子2模拟表位,为探索乳腺癌的防治研究创造了条件。     

乳腺癌干细胞富集及其特异性多肽的筛选

目的：无血清培养法富集乳腺癌干细胞（breast cancer stem cell,BCSC）并采用噬菌体展示技术,筛选能特异性结合乳腺癌干细胞的噬菌体多肽。方法：无血清培养法富集乳腺癌MDA—MB-231细胞株中干细胞并以此为靶标,以hs578bst人正常乳腺细胞及普通培养的MDA—MB-231细胞为减性筛选细胞,对噬菌体随机肽库进行双重减性筛选,选取富集后的阳性噬菌体单克隆,ELISA及DAB染色鉴定阳性噬菌体特异性并测序。结果：经过3轮筛选,噬菌体得到约500倍的富集,随机挑选10株单克隆噬菌体。ELISA显示,6号噬菌体单克隆对乳腺癌干细胞的亲和力是对照的6．14倍;DAB鉴定亦显示,其对乳腺癌干细胞的特异性及亲和力最高,对阳性噬菌体DNA测序翻译得到十二肽氨基酸为GYSASRsTIPGK。结论：通过干细胞富集及噬菌体展示技术,成功筛选出能够特异性结合乳腺癌干细胞的特异性噬菌体多肽,为乳腺癌的干细胞靶向治疗和深入研究奠定基础。     

Analysis of BAC<Subscript>5</Subscript> mcAb-related epitope using random peptide library

Objective To identify epitope relating to BAC₅ mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC₅ mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC₅ mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of “-P-V-” structure existed near N-terminus of the 3 different peptides, i. e.-H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8)-Q-N-Q-A-W-F-S-Q-P-V-R-M-(3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide “-N-H-Q-S-T-F-W-Q-K-W-T-A-” displayed by M13 phages from the negative clones (6/6). Conclusion BAC₅ mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC₅ mcAb. This project was founded by Health Foundation of Guangdong Province (Grant No. A1999212)     

Development of an enzyme-linked immunosorbent assay (ELISA) for detecting IgA antibodies to the Epstein-Barr virus

A 3-step enzyme-linked immunosorbent assay (ELISA) was developed for detecting IgA antibodies to purified Epstein-Barr virus (EBV) polypeptides. The 3-step procedure included the use of a mouse anti-human IgA monoclonal antibody (MAb) to amplify the IgA reaction. The 2 major EBV proteins used in this assay were the 125-kDa component (gp125) associated with the viral capsid antigen (VCA) complex and a major glycoprotein (gp250/200) associated with the membrane antigen (MA) complex. Eighty-two sera were tested on ELISA plates containing either both of the glycoproteins or each one separately. These included 45 IgA antibody-positive sera from patients with nasopharyngeal carcinoma (NPC). With these sera, there was a good correlation, both qualitatively and quantitatively, between results with the immunofluorescence (IF) and ELISA procedures. Although most IgA antibody-positive sera contained antibodies reactive with both gp125 and gp250/200, a number of sera contained antibodies reactive with one of the glycoproteins but not with both. The data indicated that both of these glycoproteins should be used in assays for detecting IgA antibodies to EBV, to avoid false-negative results. This assay should be useful for screening large populations for IgA antibodies to EBV and also possibly for monitoring disease course in patients with NPC.     

Serum and salivary IgA antibodies against a defined epitope of the Epstein-Barr virus nuclear antigen (EBNA) are elevated in nasopharyngeal carcinoma.

The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.     

鼻咽癌人源抗独特型单链抗体的制备及筛选

背景与目的:抗独特型抗体作为肿瘤抗原替代物可用于肿瘤治疗,这已在临床试验中得到证实。但由于目前所使用的抗独特型抗体多为鼠源性,用于人体可产生人抗鼠抗体反应,从而影响疗效。本实验拟构建噬菌体人源抗独特型抗体库,并从中筛选出能模拟鼻咽癌相关抗原的β型抗独特型单链抗体scFv(Ab2βscFv),以解决鼠源性抗独特型抗体用于临床所产生的人抗鼠抗体反应。方法:体外致敏并用EB病毒(Epstein-Barrvirus,EBV)转化鼻咽癌患者的外周血单个核细胞(peripheralbloodmononuclearcell,PBMC),用RT-PCR分别扩增VH和VL基因并连接成scFv基因,将scFv基因与载体fUSE5连接后,转化大肠杆菌MC1061,构建噬菌体呈现型scFv库。在用单抗FC2对文库进行4轮筛选后,用SandwichELISA和结合抑制法从中筛选出β型Ab2scFv。结果:用单抗FC2体外致敏并经EBV转化的10例鼻咽癌患者的PBMC中,8例有鼻咽癌抗独特型抗体产生。经PCR分别扩增出5种VH(γ、μ)和7种VL(κ、λ)基因,经连接组成14种scFv基因。在与载体连接后,导入大肠杆菌MC1061,得到库容为1.5×108的初级噬菌体抗独特型抗体库。经富集筛选后,从中随机挑取270个克隆进行ELISA筛选,得到91个Ab2scFv单克隆,阳性率为33.7%。再用结合抑制法从中初步筛选出5个可能为β型的Ab2scFv。结论:联     

噬菌体抗原模拟表位的免疫原性研究

目的 通过研究胃癌相关抗原的短肽模拟表位的免疫原性,探讨肿瘤抗原表位的噬菌体模拟肽是否具有免疫原性,为进一步的胃癌疫苗研制奠定实验基础。方法 以胃癌单克隆抗体MG7所识别的胃癌相关抗原表位模拟肽为免疫原,免疫Balb/c小鼠,获得免疫血清;用表达MG7单抗所识别的相应抗原的胃癌组织切片,通过免疫组化方法筛选免疫血清;在此基础上,对阳性血清进一步用细胞荧光标记法和ELISA法进行了检测,结果 免疫血清经免疫组化筛选和细胞荧光标记法及ELISA法检测,发现其中一个噬菌体呈现的表位模拟肽的免疫血清可与人胃癌组织间呈现特异的免疫反应,由此筛选出具有免疫原性的胃癌相关抗原表位的噬菌体模拟肽。结论 用单克隆抗体从随机肽库中筛选得到的部分表位模拟肽具有引发识别厮的始抗原免疫反应的免疫原性,提示以肿瘤相关抗原的表位模拟肽为基础研制疫菌具有可行性,可望在机体内诱发有效的体液免疫反应。     

Association of Epstein-Barr virus with nasopharyngeal carcinoma in Alaskan native patients: serum antibodies and tissue EBNA and DNA

Biopsy specimens from Alaskan Native patients with nasopharyngeal carcinoma (NPC) and from other patients seen on the otolaryngology service were tested for Epstein-Barr virus-specific DNA and nuclear antigen (EBNA). Serum samples from both groups were tested for various EBV-related antibodies. EBV DNA and EBNA results were in agreement in 29 of 31 tissue specimens tested by the two methods. Ten of 11 biopsies containing NPC cells were positive for EBV DNA. Two NPC patients had biopsies that showed only atypical epithelium but were also positive for EBV DNA or EBNA. The other tissue specimens were negative except for biopsies from two patients: one with a parotid gland lymphoepithelial lesion; another with undifferentiated carcinoma of salivary gland origin.     

Antibody response to Epstein-Barr virus Rta protein in patients with nasopharyngeal carcinoma: a new serologic parameter for diagnosis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is associated closely with Epstein-Barr virus (EBV). The authors previously reported that an EBV immediate-early gene, BRLF1, was expressed frequently in NPC tumors, and a significant elevation in immunoglobulin G (IgG) antibodies directed against BRLF1 gene product Rta was detected in NPC sera by a radioactive immunoprecipitation assay. To simplify and to make the detection more quantitative, an enzyme-linked immnunosorbent assay (ELISA) was developed in this study. METHODS: Antigen domains of Rta were identified further using an immunoprecipitation assay. Two glutathione-S-transferase (GST) recombinant Rta fragments (R150-GST and R185-GST) were prepared subsequently and were used as antigens in the ELISA. Serum samples derived from 51 patients with NPC patients, 115 non-NPC ENT patients, and 47 healthy volunteers were examined for the presence of antibodies directed against Rta. RESULTS: Among the patients with NPC, 74.5% showed a positive IgG response to R150-GST, and 62.7% showed a positive IgG response to R185-GST, with 80.4% positive for either fragment. In contrast, the reactions were positive in only 8.5% of healthy volunteers and 13.0% of control patients. When using a mixture of the two recombinant Rta proteins as coating antigens, the IgG positive responses were 82.3%, 10.6%, and 14.8%, respectively, in patients with NPC, healthy volunteers, and control patients. It is noteworthy that 51.0% of the NPC sera showed a positive immunoglobulin A (IgA) response, with none of the control patients showing obvious reactivity. Both the IgG response and the IgA response to Rta protein in patients with NPC were correlated with the IgA response to EBV early antigens and virus capsid antigens, the classic serologic markers used to diagnose NPC. CONCLUSIONS: The ELISA method described for the detection of IgG antibodies directed against recombinant Rta proteins is simple and reliable and may be useful as a serologic parameter for the screening and diagnosis of patients with NPC.