重组毕赤酵母菌高密度表达人源抗-HBs Fab抗体的研究

目的探讨抗-HBsFab抗体发酵工程菌GS115／Fab的高密度发酵及对发酵产物的纯化与活性鉴定方法。方法采用补料分批培养方法,在5L发酵罐中对发酵工程菌GS115／Fab进行高密度发酵,发酵温度28～30℃,pH值范围为5．0～5．5,溶氧范围20％以上;3次发酵分别于OD600值达到400～450、200～250、300～350时开始诱导,甲醇诱导浓度为0．5％～1％,经96h左右的诱导后终止发酵,对发酵上清进行亲和纯化,并通过ELISA法对纯化产物进行活性鉴定。结果在OD600为300～350时开始诱导有利于发酵过程条件的控制和目的蛋白的表达,目的蛋白表达量为245mg／L。发酵上清通过亲和层析纯化,获得纯度为98％的重组Fab抗体,该抗体经ELISA分析具有较高的HBsAg抗原亲和力及特异性。结论Fab酵母菌工程菌在5L发酵罐高密度发酵成功,为抗-HBsFab抗体的产业化生产及临床研究奠定了基础。     

抗Erbin特异性单克隆抗体的制备及鉴定

目的:制备抗Erbin单克隆抗体.方法:应用基因工程技术构建含Erbin PDZ序列的原核表达载体,转化大肠杆菌BL21(DE3)以获得重组蛋白的表达;采用镍离子亲和层析柱纯化重组蛋白;通过质谱鉴定重组蛋白;利用纯化的重组蛋白免疫小鼠,通过细胞融合技术制备单克隆抗体.结果:构建了原核表达载体pET-28a(+)-Erbin PDZ,获得了重组蛋白在工程菌中的高效表达.通过对以包涵体形式存在的重组蛋白进行洗涤、溶解、复性和纯化,获得了纯度在90%以上的重组蛋白.经质谱鉴定确证表达的重组蛋白为Erbin PDZ.用重组蛋白免疫小鼠后,经细胞融合、筛选及鉴定,获得了高效价的单克隆抗体.讨论:该抗体可识别天然状态下的Erbin蛋白,可用于ELISA、免疫荧光和免疫沉淀实验.抗Erbin单克隆抗体的制备为研究Erbin的功能奠定了基础.     

重组人抗-HBs单链抗体与白细胞介素2融合蛋白的表达、纯化及活性鉴定

目的 探讨抗-HBs单链抗体与白细胞介素2融合蛋白的表达、纯化及活性鉴定方法。方法 将构建的目的蛋白表达工程菌M15[pQE-ScFv-IL-2]经IPTG诱导后,通过SDS-PAGE及Western blot分析鉴定表达产物;再通过Ni^2 离子金属螯合亲和层析和离子交换层析纯化目的蛋白;采用逐步透析法对纯化后的目的蛋白进行复性后,用间接ELISA实验和CTL-2细胞增殖反应实验对其进行活性鉴定。结果 SDS-PAGE及Western blot分析结果显示有分子量约为43kD的重组蛋白表达,表达量可达18％;两步纯化后凝胶成像分析目的蛋白的纯度达到95％;复性后纯化产物的活性鉴定结果表明,重组抗体融合蛋白既能与HBsAg特异性结合,也能刺激CTLL-2细胞增殖。结论 获得的抗体融合蛋白兼具亲本分子的双特异性,为慢性乙型肝炎及相关疾病的导向治疗研究打下了良好基础。     

三种方法纯化生物工程抗体片段的比较

比较3种不同纯化体系纯化生物工程抗－HBsFab的效率和效果，寻求高效，经济的生物工程产品批量纯化方法，采用增菌发酵抗－HBsFab阳性克隆，超声破菌法制备Fab上清，分别缓冲平衡抗Fab -Sepharose亲合胶，链球菌蛋白G（prot.G)-Sepharose胶和Ni-NTA离子螯合胶，对Fab上清进行过柱纯化，紫外光检测目的蛋白得率，SDS－PAGE鉴定产物的纯度并用HBsAg包被ELISA检测其生物活性，结果显示，等容积不同类别的胶体对目的蛋白的纯化效率依次为：Ni-NTA离子螯合胶＞prot.G-Sepharose胶＞抗－FabSepharose亲合胶；在各胶体饱和结合能力之内，3种方法中获高纯度产品，在SDS－PAGE中呈现单一区带：HBsAg包被ELISA检测结果为3种方法纯化制备的Fab在抗原结合能力上未显示明显差别，提示，3种纯化方法各有优势和应用限制，Ni-NTA法在经济性，实用性和纯化效率上明显优于其他两种方法。     

半乳糖凝集素-1的表达纯化与生物活性分析

目的：获得重组人半乳糖凝集素-1（galectin-1）蛋白,并分析其生物学活性。方法：PCR方法扩增目的基因,将其克隆到原核表达载体pET21a（＋）,获得重组质粒pET-galectin;将其转化至表达宿主菌BL21（DE3）中,用IPTG进行诱导,获得目的蛋白的表达;采用Ni-NTA亲和层析柱对目的蛋白进行纯化,用红细胞凝集试验分析其生物学活性。结果与结论：成功构建重组原核表达质粒pET-galectin,目的蛋白在大肠杆菌中获得高效表达,经Ni-NTA亲和层析柱一步纯化后,得到的蛋白纯度可达80%以上。红细胞凝集试验结果表明获得的重组蛋白具有较好的生物学活性,可进一步用于半乳糖凝集素-1功能研究。     

小鼠及人源化鼠抗人交联纤维蛋白单链抗体的表达与纯化

目的：表达并纯化小鼠及人源化鼠抗人交联纤维蛋白单链抗体,并初步分析比较两者的体外生物活性。方法;经ＤＮＡ重组的构建重组表达质粒,经ＩＰＴＧ诱导,在大肠杆菌中高表达两种单链抗体,用８ｍｏｌ／Ｌ尿素溶解包涵体后经ＩＭＡＣ纯化表达产物,并用凝血酶切除Ｎ端融合的（Ｈｉｓ）６,纯化的表达产物经复性后ＥＬＩＳＡ检测其活性,结果：两种单链抗体在大肠杆菌中表达量占全菌蛋白的５０％以上,经变性,纯化后纯度可达９７％     

咪唑啉受体抗血清选择性蛋白的表达、纯化及单克隆抗体制备

目的:表达和纯化咪唑啉受体抗血清选择性蛋白(imidazoline receptor antiserum-selected protein,IRAS protein),制备IRAS蛋白的单克隆抗体.方法:采用基因重组技术在大肠杆菌表达IRAS蛋白;金属镍螯合的Ni-NTA亲和层析柱进行蛋白纯化;杂交瘤技术建立分泌IRAS单克隆抗体的杂交瘤细胞株;以间接ELISA方法筛选分泌特异性IRAS单克隆抗体的杂交瘤细胞;采用蛋白免疫印迹、间接免疫荧光方法鉴定单克隆抗体的特异性.结果:成功表达并纯化了IRAS重组蛋白,纯度达到95%.共筛选出5株分泌IRAS单克隆抗体的杂交瘤细胞株,制备腹水并纯化了IRAS的单克隆抗体,腹水中单克隆抗体效价分别为1∶ 8×106,1∶ 2×106和1∶ 5×106,属于IgG1亚型.该抗体能与原核及真核系统表达的IRAS重组蛋白发生特异性反应,间接免疫荧光显示IRAS蛋白主要定位于细胞质中.结论:建立了稳定分泌IRAS单克隆抗体的杂交瘤细胞株,成功制备了特异性好的IRAS单克隆抗体,为研究IRAS的功能提供了有力的研究工具.     

抗大鼠HSP70蛋白单克隆抗体的制备及特性研究

目的制备大鼠HSP70蛋白单克隆抗体并对其性质进行研究。方法利用PCR技术扩增大鼠全长hsp70,插入表达质粒pET-28a,pMAL-c2x,诱导表达,采用亲和层析纯化融合蛋白;用HIS-HSP70融合蛋白免疫BALB/c小鼠,杂交瘤技术制备大鼠HSP70单克隆抗体;采用Western印迹和免疫组化方法对抗体性质进行鉴定,ELISA方法确定抗体的表型。结果亲和层析纯化HIS-HSP70蛋白纯度在97.6%,获得6株可稳定分泌抗HSP70mAb的杂交瘤细胞系;抗体的亚类均是IgG1类κ链的。Western印迹结果显示6株单抗均能识别天然的大鼠HSP70蛋白;免疫组化结果显示3株抗体可以识别组织的HSP70蛋白。结论成功获得6株有活性HSP70单克隆抗体,可以识别HSP70蛋白不同表位,为进一步研究HSP70单克隆抗体在疾病中的作用奠定基础。     

幽门螺杆菌热休克蛋白A与尿素酶B融合表达产物的初步纯化及其在小鼠体内免疫效应活性分析

目的 :探讨幽门螺杆菌热休克蛋白A与尿素酶B融合蛋白的免疫生物学活性。方法 :采用亲和层析、离子交换和凝胶过滤对幽门螺杆菌热休克蛋白A与尿素酶B融合蛋白进行初步纯化 ,用粗纯化蛋白对小鼠进行免疫 ,然后分析其免疫活性。结果 :采用亲和层析或离子交换和凝胶过滤后 ,融合蛋白的纯度分别可达到 6 5 .6 %和90 .2 %。该融合蛋白可诱导小鼠产生抗幽门螺杆菌的胃肠黏液IgA和血清IgG抗体 ,并促使免疫小鼠脾脏T淋巴细胞CD4 CD8 比值的增加。结论 :重组热休克蛋白A与尿素酶B融合蛋白可以诱导在未来免疫预防中起积极作用的免疫应答反应     

用pProEXHT载体进行人FLT3配体基因的表达与产物的亲和层析纯化

目的 :探讨寡聚组氨酸多肽与目标蛋白融合基因的表达及其产物的金属离子螯合亲和层析纯化方法。方法 :将人FLT3配体 (FL)胞外段cDNA连入 pProEXHT载体 ,转入大肠杆菌实现表达。分离包涵体并进行复性处理后 ,通过Ni2 离子螯合亲和层析纯化融合蛋白表达产物。结果和结论 :6×组氨酸 FL融合蛋白的表达量约占菌体蛋白总量的 15% ,用Ni2 离子螯合亲和层析纯化表达产物 ,一次过柱的纯度可达 90 %以上 ,操作程序简便省时 ,是日后FL基因工程产品大规模制备的可行途径     

Comparative imaging and biodistribution studies with an anti-CEA monoclonal antibody and its F(ab)2 and Fab fragments in mice with colon carcinoma xenografts

An IgG1 mouse monoclonal antibody directed against CEA has been digested with papain to yield F(ab)2 and Fab fragments. Following radioiodination, intact antibody and fragments showed specific binding to cells of a CEA-producing tumour, although the immune reactivities of the fragments were lower than that of intact antibody. Gamma scintigraphy of nude mice bearing CEA-producing human tumour xenografts and injected with 131I-labelled fragments showed earlier and superior imaging of tumours than did 131I-intact antibody, and this was most marked with the Fab fragment. Sequential dissection analyses showed that this was due to earlier and higher tumour-to-blood ratios with fragments than with intact antibody, but in absolute terms the degree of localization of both fragment types was significantly lower than that of intact antibody.     

Thrombus imaging with an I-123-labeled F(ab')2 fragment of an anti- human fibrin monoclonal antibody in a rabbit model

The murine monoclonal antibody MA-15C5 binds to cross-linked fibrin but not to fibrinogen. Fab or F(ab')2 fragments of MA-15C5, labeled with iodine-123, were injected intravenously into nine rabbits with a nonocclusive 0.2-mL human plasma clot in the jugular vein and into eight control rabbits. Scintigrams obtained at hourly intervals after injection of the F(ab')2 fragment revealed a significantly higher accumulation of tracer in the clot region than in the contralateral region. The relative excess of tracer in the jugular vein region was significantly greater in the study animals than in the control animals throughout the study period. Blind reading of serial scans revealed no visible tracer accumulation in seven of eight control rabbits, whereas the scans of seven of nine study rabbits showed clearly visible accumulation (sensitivity = 77%, specificity = 87%). The Fab fragment of MA-15C5 had a lower affinity and bound less avidly to the blood clot in vivo. These results suggest that the F(ab')2 fragment has better imaging properties than the Fab fragment and that it may be useful for imaging blood clots in man.     

人白细胞介素17的多克隆抗体制备与单抗杂瘤的筛选

目的：制备重组人IL－17的兔抗血清并纯化,克隆化筛选IL－17的阳性杂交瘤克隆。方法：应用纯化的IL－17程序免疫家兔,得到兔抗人IL－17的抗血清;采用盐析法和亲和层析法对兔抗血清进行纯化,应用纯化的IL－17免疫小鼠,将其脾脏与骨髓瘤细胞SP2／0融合,采用有限衡释法和克隆化筛选阳性杂交瘤克隆,抗体的检测分别用脂糖双向扩散试验法和酶联免疫测定法（ELISA）。结果和结论：纯化得到多克隆抗体2．5ml,IgG纯度大于95％,初步得到5个阳性杂交瘤克隆,可用于以后的深入研究。     

Improved radioimaging and tumor localization with monoclonal F(ab')2

Monoclonal anti-tumor antibodies have great promise for radioimmunodetection and localization of tumors. Fab and F(ab')2 fragments, which lack the Fc fragment of antibody (Ab), are cleared more rapidly from the circulation and may have less nonspecific tissue binding than intact Ab. In radioimaging studies using a murine monoclonal antibody to carcinoembryonic antigen in a human colon carcinoma xenografted into hamsters, F(ab')2 fragments were shown superior to Fab fragments and intact antibody for scintiscanning. In double-label experiments with anti-CEA antibody and control monoclonal IgG, F(ab')2 fragments were found to give better and more rapid specific tumor localization than intact antibody or Fab fragments. F(ab')2 fragments offer significant promise for tumor imaging and possibly therapy.     

Comparative imaging and biodistribution studies with an anti-CEA monoclonal antibody and its F(ab)2 and Fab fragments in mice with colon carcinoma xenografts

An IgG₁ mouse monoclonal antibody directed against CEA has been digested with papain to yield F(ab)₂ and Fab fragments. Following radioiodination, intact antibody and fragments showed specific binding to cells of a CEA-producing tumour, although the immune reactivities of the fragments were lower than that of intact antibody. Gamma scintigraphy of nude mice bearing CEA-producing human tumour xenografts and injected with ¹³¹I-labelled fragments showed earlier and superior imaging of tumours than did ¹³¹I-intact antibody, and this was most marked with the Fab fragment. Sequential dissection analyses showed that this was due to earlier and higher tumour-to-blood ratios with fragments than with intact antibody, but in absolute terms the degree of localization of both fragment types was significantly lower than that of intact antibody.     

非竞争性ELISA法测定人源抗HBsAg Fab功能性亲和常数

目的　测定完全人源化基因工程抗体HBsAgFab的亲和常数。方法　采用非竞争性ELISA固相法 ,经确定最佳抗原包板浓度、最佳抗原包板时间及最佳抗原与抗体结合反应时间后 ,得到了HBsAg与抗体片段抗HBsAgFab及完整抗体抗HBsAgIgG的抗原抗体结合反应曲线 ,计算出抗HBsAgFab及抗HBsAgIgG的亲和常数。结果　人源基因工程抗体抗HBsAgFab的功能性亲和常数在 10 7～10 8M 1水平 ,比完整抗HBsAgIgG仅仅小约 1个数量级 (10 8～ 10 9M 1)。结论　该基因工程抗体与抗原结合能力较强 ,为今后开发应用Fab进行生物导向治疗提供了理论基础。     

Blood and tissue kinetics of radiolabelled anti-CEA monoclonal antibody and F(ab)2 and Fab fragments in nude mice with human tumour xenografts: implications for tumour imaging and radioimmunotherapy

The kinetics of a radiolabelled anti-CEA monoclonal antibody and its F(ab)2 and Fab fragments have been examined in mice including those with human tumour xenografts reactive with this antibody. With intact antibody the blood alpha phase half time was 9.9 h, the beta catabolic phase 128 h, with 55% of the remaining antibody in the intravascular compartment. Tumour localization was slow, peak levels of 22% of the dose g-1 being attained by 24 h, but remaining constant to at least 48 h. F(ab)2 fragments alpha and beta half times were 2.95 and 7.8 h, with an intravascular fraction of only 11%. Peak levels of tumour localization were seen within 7 h of injection but reached only 10% of the dose g-1 and then declined. Fab fragment half times were only 0.31 and 5.4 h, with 9% remaining intravascularly. Tumour localization was seen early but at only 2.5% of the dose g-1, and then declined. Tumour to blood ratios increased over time with all preparations, but reached only 2.5:1 with intact antibody compared with 15:1 and 19:1 with F(ab)2 and Fab respectively. These studies emphasize that the improved tumour: normal tissue ratios obtained with antibody fragments are the result of both faster and greater extravasation of fragments and their faster overall catabolism.     

Tumor location using F(ab')2 mu from a monoclonal IgM antibody: pharmacokinetics

A monoclonal IgM antibody (anti-SSEA-1) and its divalent antigen-binding peptic fragment [F(ab')2 mu] were compared as in vivo tumor localization reagents in mouse teratocarcinomas. F(ab')2 mu is cleared more rapidly than whole antibody from the whole body, blood, and all tested organs (t1/2 for whole antibody approximately 18 hr; t1/2 for F(ab')2 mu, 12 hr). A corresponding average improvement in tumor-to-tissue ratio is observed 48 hr after injection and earlier. However, the affinity of the F(ab')2 mu for antigen is much lower, and a smaller fraction of the antibody fragment is retained in the tumor than with whole antibody. The fragment was not retained by animals bearing nonantigenic tumors.     

住友CLC模块在线优化合成^13N-NH3· H2O的研究

目的 确定住友HM-10回旋加速器相关参数,小步优化住友CLC模块,合成显像良好的^13N-NH3· H2O.方法 优化住友HM-10回旋加速器的束流大小、轰靶时间和靶水中乙醇去自由基含量,提高化学反应效率.用阳离子交换柱（CM柱）吸附靶水和小步优化纯化流程得到高化学纯度的^13N-NH3·H2O,并实现一根CM柱上多次吸附纯化（3次左右）.结果 用30 μA束流轰击10 mmol/L乙醇11 min,合成^13N-NH3·H2O 27批次,产量为925 MBq左右,放化纯度和化学纯度均大于99％,注射大狗后心肌灌注显像良好.结论 经过回旋加速器锂靶反应条件的优化和住友CLC纯化模块的小步改进能得到产量稳定、显像良好的^13N-NH3· H2O,能够满足实验及临床要求.     

人斯钙素1在大肠杆菌中的重组表达与活性鉴定

目的：通过原核表达的方法得到有活性的斯钙素1（ STC1）重组蛋白。方法将优化合成的STC1 DNA片段克隆到表达载体pET-32 b（＋）上,重组质粒转入大肠杆菌诱导表达,在变性条件下纯化包涵体,经透析复性得到可溶的斯钙素1融合蛋白,凝血酶酶切后用高亲和镍离子树脂吸附多余多肽获得目的蛋白。 Western印迹分析验证目的蛋白免疫活性。动物实验检测目的蛋白的生物学活性。结果构建了pET-32b（＋）-STC1表达载体,表达并纯化了STC1融合蛋白包涵体,经复性、凝血酶切和纯化后获得可溶的目的蛋白。 Western印迹分析验证正确。生物学活性检测表明重组STC1能增加大鼠排泄系统对磷酸盐的重吸收。结论获得了具有生物学活性的斯钙素1重组蛋白,为制备STC1单克隆抗体和进一步研究STC1与肿瘤治疗的关系奠定了基础。