抗内毒素噬菌体抗体库的构建及筛选

目的构建一个鼠源性的抗内毒素单链噬菌体抗体库,从中筛选出对内毒素具有较高亲和力的单链抗体。方法从小鼠脾细胞中提取总RNA,通过RT-PCR技术扩增出小鼠抗体重链、轻链可变区基因（VH,VL）,用Linker将VH,VL交联形成单链抗体可变区片段（ScFv）。经NotⅠ,SfiⅠ双酶切后与经同样双酶切的pCANTAB5E载体相连,转化入大肠杆菌TG1以构建鼠抗内毒素单链噬菌体抗体库。在援救噬菌体抗体库后,用内毒素淘筛特异性的ScFv,富集的噬菌体阳性克隆重新感染TG1。在96孔板分别援救单个含特异性ScFv的TG1菌落,最后随机挑选出190个菌落经ELISA检测抗内毒素ScFv。结果小鼠血清中抗内毒素的效价为1∶12800。提取的总RNA浓度为12.3813μg/ml,纯度较好。扩增出的VH长约340bp,VL约320bp,ScFv约800bp。转化入TG1后有约1.9×107个菌落。淘筛一轮过后即有3×104阳性菌落长出,190个菌落经ELISA检测有2个阳性克隆。结论成功地构建了一个库容量为1.9×107的鼠抗内毒素单链噬菌体抗体库,并从中筛选出了2株抗内毒素ScFv。     

抗γ-精浆蛋白人-鼠杂合Fab噬菌体抗体库的构建及导向筛选

目的:建立人-鼠杂合Fab噬菌体抗体库,筛选抗γ-精浆蛋白(γ-sm)人源性抗体轻链。方法:PCR扩增前列腺癌患者外周血淋巴细胞人抗体全套轻链基因,克隆人含鼠源抗γ-sm抗体Fd基因的pComb3X噬粒中,建立人-鼠杂合Fab噬菌体抗体库。通过稀释滴定、限制性酶切等分别对库容、基因重组率和多样性进行鉴定。以M13K07辅助噬菌体超感染,利用纯化的γ-sm对杂合库进行筛选,对筛出的阳性克隆进行功能检测。结果:构建了库容量为1．2×107CFU、重组率为90％、多样性好的人-鼠杂合噬菌体抗体库。ELISA检测出2个强阳性克隆,Western blot确证为抗γ--sm的人-鼠杂合Fab,竞争ELISA显示其表观亲和力约为亲本鼠抗体亲和力的71．8％。DNA测序显示筛到的人源轻链可变区基因属IGKV4-1*01胚系家族。结论:成功得到人源性抗γ-sm抗体轻链,为进一步获得全人源化抗体奠定了基础。     

全人源食管癌噬菌体单链抗体库的筛选及特异性鉴定

目的：我们应用噬菌体抗体库技术构建了食管癌相关的人源单链抗体文库，本文目的在于对该抗体库进行鉴定，筛选食管癌抗体，同时对抗体的活性进行检测。方法：PCR鉴定TG1中食管癌单链抗体scFv的插入率；1．5％琼脂糖凝胶电泳鉴定Sfi I和NotI双酶切质粒的结果；先以人正常食管上皮细胞吸附后再以食管癌细胞Eca109为抗原对所建抗体库进行4轮的亲和筛选；将阳性重组噬菌体克隆感染Ecoli HB2151进行可溶性抗体表达及经亲和柱层析纯化；用SDS—PAGE测定该抗体的相对分子量；用Western blot鉴定该抗体；用ELISA法、免疫组化法鉴定该抗体与人食管癌细胞的结合的特异性。结果：seFv基因插入率为91．7％；酶切后检测到目的基因片段。在亲和筛选过程中，食管癌噬菌体单链抗体得到富集，收获率逐轮得到提高，第4轮为第1轮的141倍；SDS—PAGE与Western blot结果显示抗体的相对分子量为左右和30kd条带染色；在Ecoli HB2151中实现了单链抗体的可溶性表达。免疫组织化学结果提示可溶性抗体仅染食管癌组织，而肝癌组织和胃癌组织不染色。免疫细胞化学结果表明此可溶性抗体可使Eca109细胞染色。ELISA测定结果显示可溶性抗体具有较高的免疫活性，能与Eca109细胞结合，而不与胃癌BGC-823和NHEEC结合。结论：利用噬菌体抗体库技术的阴性、阳性筛选得到了食管癌噬菌体单链抗体，且筛选后的抗体片段与人食管癌细胞有特异性的结合活性。     

全人源抗乳腺癌噬菌体单链抗体库的构建

目的:利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法:从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞(PBMC),提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链(VH)和轻链(VL)基因,经重叠延伸PCR(SOE-PCR),在体外将两者连接成单链抗体(scFv)基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果:得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论:本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。     

全人源抗乳腺癌噬菌体单链抗体库的构建

目的：利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法：从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞（PBMC）,提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链（VH）和轻链（VL）基因,经重叠延伸PCR（SOE-PCR）,在体外将两者连接成单链抗体（scFv）基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果：得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论：本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。     

抗前列腺癌细胞特异抗体库的构建及特异结合抗体的筛选

目的:研究复方中药VI-28胶囊(即卫康胶囊,主要成分有人参、鹿茸、菟丝子)辅助环磷酰胺体内抑制肿瘤生长的作用及其机制。方法:C57BL/6纯系小鼠背部皮下接种Lewis肺癌细胞(LCC,每只106个细胞),同时按40mg/kg隔天腹腔注射环磷酰胺治疗。实验组灌胃VI-28(2%悬液,每只0.5ml,隔天给药)。第28天时取小鼠胸腺和肿瘤组织块,计算肿瘤指数和胸腺指数。取脾细胞测定细胞免疫功能。组织切片观察所形成实体瘤的病理学特征。结果:小鼠接种LLC细胞14d后在接种部位开始形成实体瘤,瘤块直径增加为1—1.8cm。与单纯化疗荷瘤组小鼠相比,VI-28辅助治疗组的胸腺较大,脾细胞对ConA反应性较强,形成实体瘤的瘤块较小且分化程度较高。结论:VI-28胶囊可有效地促进化疗小鼠免疫功能的恢复,帮助化疗药物抑制肿瘤生长,是一种有效的化疗辅助药。     

全人源肝癌噬菌体单链抗体的筛选及特异性鉴定

目的：对全人源肝癌噬菌体单链抗体库进行鉴定，筛选肝癌抗体，同时对抗体的活性及特异性进行鉴定。方法：PCR鉴定阳性重组菌TG1中人肝癌ScFv的插入率。先以人成纤维细胞吸附后再以体外培养的肝癌细胞SMMC-7721为抗原对所建抗体库进行3轮“吸附-洗脱-扩增”的亲和筛选。将筛选后的ScFv进行PCR鉴定及双酶切鉴定；通过ELISA法及FCM鉴定其与人肝癌细胞及正常细胞的结合活性。结果：ScFv基因插入率为70％。在亲和筛选过程中，肝癌噬菌体单链抗体得到富集，收获率逐轮得到提高，第3轮为第1轮的214倍。筛选后的ScFv进行PCR鉴定及双酶切鉴定，均可检测到目的基因。ELISA分析结果显示18个克隆与SMMC-7721呈阳性反应，阳性率为90％，15个克隆与成纤维细胞有交叉反应。得到3株肝癌单链抗体。ScFv的FCM鉴定表明，以正常胎肝细胞L-02为对照，ScFv与肝癌结合比率为41．3％。特异性鉴定表明，其与肝癌细胞结合活性明显高于正常细胞。结论：利用噬菌体抗体库技术结合减数筛选得到了肝癌噬菌体单链抗体及其基因，且筛选后的抗体片段与人肝癌细胞有特异性的结合活性。     

用不同方法从大容量噬菌体抗体库中筛选抗完整食管癌细胞的单链抗体

目的:初步探索从天然的单链大容量噬菌体抗体库中筛选肿瘤细胞抗体的技术策略.方法:通过两种方法,即2%多聚甲醛固定细胞筛选法(PF)及活细胞直接筛选法(NF),经过4轮"吸附-洗脱-扩增"的淘筛过程,对食管癌KYSE-170细胞进行筛选,得到抗食管癌细胞的噬菌体抗体,完善了噬菌体抗体库筛选完整细胞的特异性抗体的技术策略.并且进一步制备了其可溶性抗体.结果:活细胞NF组的筛选阳性率高于2%多聚甲醛固定PF组,筛选特异性抗体的阳性率分别为25.00%和12.50%,两组的差异具有统计学意义.结论:利用单链大容量抗体库可以采用不同的筛选方法成功制备与肿瘤细胞结合的噬菌体抗体,为今后的研究和应用奠定基础.     

噬菌体抗体库的筛选策略及其进展

噬菌体抗体库技术的出现开创了一条简便快捷的基因工程抗体生产路线,为人源抗体的制备提供了新途径.随着现代分子生物技术的迅速发展,抗体库的筛选工作也在传统的亲和淘选的基础上不断改进,涌现出越来越多新的有效筛选方法,显示出噬菌体抗体库技术在抗体开发应用中极为广阔的前景.     

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency oftraditional techniques both in diagnosis and therapy of the disease makes the development of alternative and noveltechniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be usedto increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of thisstudy is screening and identification of individual peptides specifically targeted to human gastric cancer cells usinga phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptidesequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cellswere used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide librarywere applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were establishedby enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clonesafter five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequenciesof each amino acid in best binding clones to determine positively overexpressed amino acids for designing novelpeptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific bindingon MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acidfrequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, butmore importantly, data extracted from eluted phage clones may be used to design theoretical peptides with betterbinding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potentialcandidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research.     

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。     

t—PSA,f—PSA／t—PSA诊断前列腺癌的临床意义

为了探讨游离前列腺特异抗原（ｆＰＳＡ）与总前列腺特异抗原（ｔＰＳＡ）比率对前列腺癌的诊断价值,采用酶联免疫方法分别测定４１例前列腺癌病人,３６例前列腺增生（ＢＰＨ）病人血清ｔＰＳＡ、ｆＰＳＡ,并计算出ｆＰＳＡ／ｔＰＳＡ比率。ｔＰＳＡ界限值为４μｇ／Ｌ时,敏感度,特异度,准确度分别为８２．９％,６０．０％,７２．４％;ｆＰＳＡ／ｔＰＳＡ界限值定为１７．０％时,敏感度,特异度,准确度分别为８７．８％,９３．０％,８４．２％。结果表明：ｆＰＳＡ／ｔＰＳＡ在保持敏感度的同时,可显著地提高特异度,能更有效地诊断前列腺癌     

人卵巢癌cDNA表达文库的构建

以polyA~ mRNA提取试剂盒从卵巢癌组织中获得高质量的polyA~ mRNA,并以此合成第1、2链cDNA.cDNA两端补平后加EcoR Ⅰ接头,Xbo酶切,通过分级分离去除<400bP的片段.取100ng cDNA与噬菌体表达载体Uni-ZAP XR连接,体外进行噬菌体DNA包装,并立即进行文库的滴定和扩增,获得卵巢癌cDNA表达文库.该文库原始重组子为10~6独立克隆;重组率>99%;并以引物PUCⅠ、PUCⅡ扩增插入片段,平均大小约为1.1kb.证实此cDNA表达文库合格.以PCR从此cDNA表达文库中筛选HLA-DPB和β-actin基因,获得成功.     

人源抗肝癌单链抗体库的构建及初步鉴定

目的构建人源抗肝癌单链抗体基因噬菌体表面呈现文库。方法利用噬菌体表面呈现技术,构建基因文库,经过panning筛选富集后,用ELISA方法检验抗原结合活性。结果从30个噬菌体克隆中筛选到8个具有肝癌细胞株SMMC7721结合活性的阳性克隆。结论从外周血淋巴细胞中获取可变区基因,利用噬菌体抗体库技术制备人源抗肝癌单链抗体的策略是可行的。     

Adenoid Cystic Carcinoma/Basal Cell Carcinoma of the Prostate: Overview and Update on Rare Prostate Cancer Subtypes

Adenoid cystic carcinoma/basaloid cell carcinoma of the prostate (ACC/BCC) is a very rare variant of prostate cancer with uncertain behavior. Few cases are reported in the literature. Data on treatment options are scarce. The aim of our work was to retrospectively review the published reports. Thirty-three case reports or case series were analyzed (106 patients in total). Pathological features, management, and follow-up information were evaluated. Despite the relatively low level of evidence given the unavoidable lack of prospective trials for such a rare prostate tumor, the following considerations were made: prostate ACC/BCC is an aggressive tumor often presenting with locally advanced disease and incidental diagnosis occurs during transurethral resection of the prostate for urinary obstructive symptoms. Prostate-specific antigen was not a reliable marker for diagnosis nor follow-up. Adequate staging with Computed Tomography (CT) scan and Magnetic Resonance Imaging (MRI) should be performed before treatment and during follow-up, while there is no evidence for the use of Positron Emission Tomography (PET). Radical surgery with negative margins and possibly adjuvant radiotherapy appear to be the treatments of choice. The response to androgen deprivation therapy was poor. Currently, there is no evidence of the use of truly effective systemic therapies.     

非小细胞肺癌T7噬菌体展示文库的构建

Objective: Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC) diagnosis, new biomarker, such as serum autoantibodies may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocercinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods: mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library. Results: Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5 x 1010 pfu/mL. PCR amplifica-tion of random plaque show insert ratio were 100% (24/24) in adenocarcinorna library and 95.8% in human lung squamas carcinoma library (23/24). Insert range from 300 bp to 1 500 bp. Conclusion: Two phage display cDNA library from NSCLC were constructed.