噬菌体抗体库筛选技术及在肿瘤研究中的应用

噬菌体抗体是指表达在噬菌体表面的抗体分子Fab或scFv,这些分子的群体称为抗体库。经特定抗原或细胞筛选后可以获得针对该抗原或细胞表面标志物的特异性抗体。经典的筛选方法为固相或液相抗原识别,前提条件是能得到抗原纯品,对于抗原无法提纯或抗原性质不确定的情况则不能适用,所以又出现全细胞筛选,直接利用肿瘤细胞或组织细胞进行筛选,无需纯抗原,但目标抗原一定要有较高的表达水平,对于表达较低的情况,一种策略是先利用正常细胞筛选,去掉无关抗原后,再利用肿瘤细胞筛选;另一种策略是细胞内化筛选,特异性抗体被内化入细胞,然后用酸性洗脱液洗脱掉细胞膜上非特异结合的噬菌体抗体后裂解细胞,获得特异性抗体。其余筛选方法还有切片组织筛选、体内筛选等,适用面较窄,未获推广。筛选效率主要通过以下几项的检测:转化数,即被感染的噬菌体数,它随筛选轮数的增加而增加;抗体基因插入载体的频率,高比例的丢失意味着筛选的低效率;抗体亲和性。抗体库技术避免了杂交瘤技术中的许多难点,如细胞融合、动物免疫等,目前该技术已逐渐在肿瘤诊断和肿瘤治疗中发挥重要作用。

新式SOE PCR高效组装人单链抗体在高容量人天然噬菌体抗体库中的应用

目的探讨新式SOEPCR高效组装人单链抗体在高容量人天然噬菌体抗体库中的应用。方法通过引物设计的改变,将6种人VH基因和11种人VL基因利用两步法直接互相两两连接为人scFv基因,并且与传统的SOE法进行对比。scFv基因用于构建噬菌体单链抗体库。结果新式SOEPCR法成功的将6种人VH基因和11种人VL基因连接成66种不同的人scFv基因,与传统的SOEPCR法相比,效率高且方法简便,经过约130次电转化后,抗体库的总库容为5·58×109。结论成功地改进了传统的SOEPCR方法,高效组装了66种不同的人scFv基因,提高了抗体库的多样性。

非结核分枝杆菌病的噬菌体临床治疗策略

非结核分枝杆菌(Nontuberculous mycobacteria,NTM)病已成为全球重要的公共卫生问题之一,其发病率逐年上涨,已引起越来越多的关注。分枝杆菌对抗菌药物天然或获得性耐药,使得其治疗手段十分有限,NTM病的治疗难度较结核病大,疗程长且疗效不确切,严重影响患者的生存与生活质量。近年来,能够特异性裂解细菌的噬菌体成为研究的热点。本文拟对分枝杆菌噬菌体的生物学特性、改造技术、临床治疗应用以及目前存在的问题进行阐述,以提高目前对NTM噬菌体研究及临床应用的认识。

PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

Affinity peptide developed by phage display selection for targeting gastric cancer

AIM:To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer.METHODS:A peptide screen was performed by biopanning the PhD-12 phage display library,clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues.Tumortargeted binding of selected peptides was confirmed by bound phage counts,enzyme-linked immunosorbent assay,competitive inhibition,fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues.RESULTS:Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normal-appearing gastric mucosa.After the third round of positive screening,the peptide sequence AADNAKTKSFPV(AAD) appeared in 25%(12/48) of the analyzed phages.For the control peptide,these values were 6.8 ± 2.3,5.1 ± 1.7,3.5 ± 2.1,4.6 ± 1.9 and 1.1 ± 0.5,respectively.The values for AAD peptide were statistically signif icant(P < 0.01) for gastric cancer as compared with other histological classif ications and control peptide.CONCLUSION:A novel peptide is discovered to have a specific binding activity to gastric cancer,and can be used to distinguish neoplastic from normal gastric mucosa,demonstrating the potential for early cancer detection on endoscopy.

Selection of the Mimic Epitopes of Antigens from Schistosoma japonicum by Using Phage Display Techniques

To obtain short peptides simulating antigenic epitopes related to natural resistance against Schistosoma japonicum (S.j) in rats, and to explore their immune protection against S.j in mice, phage random peptide library of 12 amino acids were screened with purified IgG from normal rat sera. Positive clones that were obtained after three rounds of biopanning were detected by ELISA, and two of them were sequenced. Kunming mice were immunized with mixed phage clones. Each mouse was challenged with 40±1 S.j cercariae, and all mice were perfused 45 days post-challenge. The worms and the liver eggs were counted. The results were that the specific phages binding to IgG were enriched 300 folds after three rounds of biopanning. Twenty clones were detected by ELISA and 19 of them bound to the specific IgG of rat sera. The sequence of two clones revealed no homology with other sequences in the GenBank. Compared with the control groups, the reduction rates of the worm burden and liver egg were 33.6% and 59.8%, respectively. It was concluded that the specific peptides, which simulate antigenic molecules correlated with natural resistance to S.j in rats could be obtained by immunosceening phage random peptide library and a protective immunity against S.j can be detected by these epitopes in mice.

青海高原喜马拉雅旱獭血清中特异性鼠疫噬菌体抗体的检测

目的利用微量法间接检测青海高原喜马拉雅旱獭血清中特异性鼠疫噬菌体抗体,为后续噬菌体和哺乳动物免疫学之间的相互作用、噬菌体治疗、噬菌体与生态学研究提供理论依据。方法以青藏高原鼠疫疫源地分离的3株野生型鼠疫噬菌体和实验室诊断用鼠疫噬菌体为抗原,利用微量板法和双层琼脂平板法定性检测青海高原同德县、贵南县、共和县、兴海县、天峻县5个疫源县采集于2020年、2021年7—9月份喜马拉雅旱獭血清中的特异性鼠疫噬菌体免疫抗体。结果4株鼠疫噬菌体分别与847份喜马拉雅旱獭血清进行中和试验,通过点滴法均未检测到与鼠疫噬菌体抗原反应的特异性噬菌体免疫抗体。结论青海高原喜马拉雅旱獭血清中未发现特异性鼠疫噬菌体抗体,这与2020—2021年青海省天峻县、同德县、共和县、兴海县、贵南县5个鼠疫疫源县采样地点的鼠疫流行病学显示为静息期无鼠疫病原体存在的流行特点一致,即无鼠疫病原体的存在,间接说明这些鼠疫疫源地鼠疫噬菌体不存在或者比较微弱的存在,故检测不到特异性鼠疫噬菌体免疫抗体。可能反映了宿主动物与鼠疫噬菌体自然接触频率少,抗噬菌体抗体可能与鼠疫感染的形式和疾病的强度有关。

全人源抗鼻咽癌噬菌体单链抗体的筛选与鉴定

目的从全人源抗鼻咽癌噬菌体抗体库中筛选特异性单链抗体（ScFv），并对其特异性进行鉴定。方法通过噬菌体表面展示技术把ScFv表达在噬菌体表面，以鼻咽癌细胞作为抗原，用抗原递减法，通过“吸附-洗脱-扩增”过程筛选并富集特异性抗体，及ELISA筛选，获得特异阳性克隆进行免疫组化鉴定并测序。结果通过对抗体库进行三轮正负淘洗和富集后，随机挑选4212个克隆进行ELISA，发现3个克隆对CNE2呈强阳性反应，而与人正常细胞系HUVEC等呈弱阳性反应或不反应。对克隆HNSA033进一步进行免疫细胞化学验证，结果与ELISA反应一致；免疫组织化学鉴定表明克隆HNSA033与鼻咽癌组织和鼻咽组织阳性率的差别有统计学意义。结论通过淘选富集、ELISA和免疫化学鉴定获得特异性较强的噬菌体克隆，为鼻咽癌发病机制的研究和临床诊断以及治疗奠定了基础。