重组人抗HBsAg Fab抗体的纯化方法比较研究

目的研究重组人抗HBsAg Fab抗体的纯化条件。方法采用羊抗人Fab抗体亲和层析柱、14F7单克隆抗体亲和层析柱、离子交换-分子筛层析柱,分别纯化由酵母工程菌(Gs115／Fab)发酵的重组人抗HBsAg Fab抗体,并对3种纯化方法所得Fab抗体的纯度、收率、与HBsAg的结合活性进行比较。结果3种纯化方法中,14W单克隆抗体柱纯化的Fab抗体的纯度达98％左右,Fab抗体柱纯化的Fab抗体的纯度为95％,但这两种亲和柱的目的蛋白收率都不高,分别为35％、55％。而离子交换柱纯化的Fab抗体的纯度为93．8％,经分子筛柱进一步纯化后,可达98％以上,Fab抗体蛋白收率可达80％以上。经ELISA分析,3种方法纯化的Fab抗体均具有较高的HBsAg抗原结合力和特异性。结论通过对3种纯化方法的比较得出,离子交换一分子筛层析法是重组人抗HBsAg Fab抗体的最佳纯化方法,这为抗HBsAg Fab抗体的产业化生产和临床研究打下良好基础。     

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。     

Ni2+亲和胶的制备及其对带His6标签融合蛋白的纯化

目的：制备Ni^2 亲和胶，并检测其纯化带His6标签融合蛋白的效果。方法：在强碱条件下，用环氧氯丙烷活化Sepharose 4B后，接上多个IDA臂，将Ni2 连接在IDA臂上，即为亲和胶成品，用亲和胶分别在变性和非变性的条件下，纯化包涵体和可溶性表达的带His6标签的人TRF1和人tankyrase的PARP结构域，并通过Western印迹鉴定纯化的靶蛋白。结果：用自制的Ni2 亲和胶可以获得SDS－PAGE单一条带的目的蛋白，并得到Western印迹的鉴定，结论：自制的Ni^2 亲和胶对带His6标签融合蛋白的纯化能力与商品胶完全等同，但价格低廉。     

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。     

抗—HIV单克隆抗体的的筛选与鉴定

我们用HIV抗体阳性者血浆包被聚苯乙烯条，加入纯化的HIV组织培养抗原，通过夹心ELISA法，筛选出3株能分泌抗HIV单克隆抗体杂交瘤细胞，传代稳定。培养上清经Westernblot染色鉴定含有抗-HIVgag蛋白，夹心ELISA法测得杂交瘤细胞培养上清中单克隆抗体滴达到1：6400，接种BALB/C小鼠获腹水抗体效价为1：2430000。实验表明，用此单克隆抗体检测HIV病人血清抗体敏感、特异、稳定、重复性好，是一种良好的试剂。     

乙型肝炎病毒多表位嵌合蛋白的表达、纯化和鉴定

目的：构建含乙肝病毒表达抗原前S1，S2（HBV preS1,preS2)优势B细胞表位与乙肝病毒核心抗原（HBcAg)的嵌合蛋白，探索其作为兼具预防和治疗HBV感染作用的新型疫苗的可能性。方法：利用分子克隆技术先后将HBV preS1(21-47AA.),preS2(133-145AA.)表位基因插入HBcAg基因中，得到HBV C144，CS1，CS1S2融合基因，分别克隆到原核表达载体pQE-30中，大肠杆菌（E.coli)M15中进行表达，用Ni^2 固相化的螯合Sepharose Fast Flow亲和层析纯化重组蛋白，最后进行抗原性的鉴定。结果：构建了HBV嵌合型颗粒蛋白表达载体，并在E.coli中高效表达出可溶性病毒样颗粒蛋白C144,CS1,CS1S2，经Ni-NTA亲和层析 化后，蛋白纯度达80％,Western印迹及ELISA证明蛋白各表位都具有抗原性。结论：本研究为进一步深入研究新型HBV治疗性疫苗的功能和应用奠定了基础。     

抗A型肉毒毒素人源单链抗体的表达、纯化及结合活性分析

目的：实现特异性抗A型肉毒毒素人源单链抗体(ScFv)的表达与纯化，并进行结合活性分析．。方法：克隆单链抗体基因ScFv(VH-Linker-Vk)，利用pET22b表达载体构建重组表达质粒，在大肠杆菌BL21(DE3)中进行IPTG化学诱导，固相亲和层析纯化蛋白，ELISA测定其特异结合活性。结果：pET22b可稳定表达人源单链抗体基因，表达产物占全茵蛋白的25％，表达蛋白全部以包涵体形式存在于胞内。经IMAC纯化，蛋白纯度大于95％。竞争性ELISA测定结果表明，重组抗毒素在体外具有良好的活性，可竞争肉毒马血清与肉毒毒素结合。结论：采用原核表达系统可实现抗A型肉毒毒素人源单链抗体的高效表达，重组人源单链抗体具有抗原特异结合活性。     

A型肉毒毒素多表位串联体的设计、表达、纯化及鉴定

目的:设计、表达两个A型肉毒毒素多表位串联体.方法:从文献报道的A型肉毒毒素(botulinum neurotoxin type A,BoNT/A)的表位及生物信息学方法预测得到的B细胞表位中遴选表位,并加入适当的辅助性元件,设计多表位串联体A、B.对其基因进行优化后,经重叠PCR方法合成串联体A、B的全长基因.分别插入原核表达载体pQE-30,再转化E.coli M15[pREP4]感受态细胞中进行诱导表达,以金属螯合亲和层析法纯化重组蛋白,并进行鉴定.结果与结论:成功设计并构建了两个多表位串联体A、B,并在E.coli中以包涵体形式获得高效表达.Ni-NTA法纯化后分别获得纯度大于92.2%、99%的重组串联体A、B蛋白,并经透析复性法复性.Western印迹和间接ELISA显示纯化、复性后的重组蛋白与抗天然BoNT/A马血清有特异性结合反应.     

丙型肝炎病毒NS5B基因的克隆及在大肠杆菌的分泌表达

由于全长丙型肝炎病毒NS5B的疏水性，其表达和纯化非常困难。为了分泌表达NS5B，作者对NS5B进行截短，缺失其疏水部分从而在大肠杆菌细胞中分泌表达HCV NS5B基因，并测定其活性。用逆转录多聚酶链反应（RT－PCR）的方法，设计截去NS5B疏水部分，PCR引物以HCV全长质粒pBRTM/HCV-1为模板，克隆到pGEM-Teasy载体中，双酶切后回收连接到pET-21b中表达。大肠杆菌培养上清过柱纯化，进行十二烷基磺酸钠－聚丙烯酰胺凝胶电泳（SDS－PAGE）和Western免疫印迹显示NS5B蛋白在大肠杆菌细胞中表达。表达产物在大肠杆菌培养上清中存在，分子量68kD左右，而且[^3H]总掺入率达6900cpm。NS5B蛋白在大肠杆菌上清中表达成功，经过活性测定，所表达的NS5B具有活性功能。     

重组肉毒毒素E(BoNT/E)重链C端的表达、纯化及其抗原性分析

目的：克隆、表达并纯化肉毒毒素E重链C端(BoNT／EHC2)保护性抗原片段，为BoNT／E的抗体制备和检测方法的建立奠定基础。方法：采用PCR扩增BoNT／EHc2基因，插入表达载体pET28a，转化E．coli BL2l(DE3)pLysS获得表达工程菌株，并对诱导表达条件和纯化条件进行优化。利用Western印迹和间接ELISA方法鉴定rBoNT／EHc2的抗原性。结果：表达工程菌pEq28a-EHC2／BL21(DE3)pLysS于37℃用0．2mmol／L IPTG诱导6h，表达的目的蛋白可以达到全茵蛋白的37．9％。经过固定化金属螯合亲和层析一步纯化，rBoNT／EHC2的纯度可达95％以上。Western印迹和间接ELISA显示其具有良好的抗原性。结论：首次克隆、表达并纯化了BoNT／EHC2片段，并可被抗天然BoNT／E马血清所识别，为制备相应的抗体及建立快速检测方法做好了准备。     

弗氏志贺菌2a 2457T株蛋白复合体BN/SDS-PAGE电泳方法的建立

目的建立分离弗氏志贺菌2a2457T株胞内可溶性复合物的BN/SDS-PAGE电泳方法 ,并优化分离条件。方法分别比较了不同配胶方式、配胶长度以及胶条平衡方法。结果成功建立了分离弗氏志贺菌2a2457T株胞内可溶性复合物的方法。结论本实验所建立及优化的方法 ,可以有效地分离蛋白复合体,为进一步研究志贺菌属蛋白复合体奠定了基础,并对以后的实验具有一定的参考价值。     

131 I标记人源抗HBs Fab瘤内注射治疗荷人肝癌裸鼠的实验研究

目的　探讨13 1I标记人源抗乙肝表面抗原单克隆抗体Fab片段 (抗HBsFab)瘤内给药治疗荷人肝癌裸鼠移植瘤的合理性。方法　荷瘤裸鼠分为 5组 ,分别经瘤内注射13 1I 抗HBsFab、13 1I 无关Fab、13 1I、PBS及腹腔注射13 1I 抗HBsFab。 5d后每组各取 2只作组织分布测定 ,其余观察 3周 ,计算各组肿瘤生长抑制率。结果　瘤内注射13 1I 抗HBsFab组放射性计数瘤 /肝比值是腹腔注射组的 9倍 ,3周后前者肿瘤生长抑制率高于后者 ,分别为 62 .3%和 46.7%。结论　采用瘤内注射13 1I标记人源抗HBsFab导向治疗肝癌 ,具有低毒高效的治疗作用 ,临床实用价值大     

骨质疏松疫苗载体:Qβ病毒样颗粒的表达、鉴定和纯化

 目的 优化表达和纯化方式,制备出具有正确空间结构且纯度和产量较高的骨质疏松疫苗载体Qβ病毒样颗粒(Qβvirus-like particles,Qβ-VLPs)。方法 在基因合成时删除A1基因序列中Qβ衣壳蛋白终止密码子之后的序列,只合成Qβ衣壳蛋白的基因序列。将Qβ衣壳蛋白基因序列克隆到pET30a载体质粒上,用BL21(DE3)、BL21(DE3)pLysS、Rosetta(DE3)三种不同菌株进行蛋白表达,然后用SDS-PAGE和透射电镜验证是否表达出Qβ-VLPs的正确结构。设置2个蛋白表达诱导剂IPTG( Isopropyl β-D-1-thiogalactopyranoside,异丙基-β-D-硫代半乳糖苷)浓度和3种菌体破碎方式,分别优化蛋白表达条件和菌体破碎方案,然后将得到的Qβ-VLPs上清液通过硫酸铵聚沉、高速离心和分子筛分选进行Qβ-VLPs的纯化。结果 仅BL21(DE3)pLysS菌株可以表达出SDS-PAGE中呈现5-6聚体、透射电镜下呈现25~30 nm球形结构的Qβ-VLPs。使用0.5 mM浓度的IPTG表达的Qβ-VLPs产量较0.2 mM时高2.8倍,使用超声破碎菌体的方式可以获得较高的蛋白分离效率。将Qβ-VLPs上清液通过本实验过程中的方案进行纯化后纯度可达90%左右。结论 该实验探索出具有正确空间结构、组成单一且纯度较高的Qβ-VLPs的制备方案,为相关疫苗制备和研究奠定基础。     

Complementary roles of antibody affinity and specificity for in vivo diagnostic cardiovascular targeting: How specific is antimyosin for irreversible myocardial damage?

BACKGROUND: Identification of irreversible myocyte injury with antimyosin antibody imaging depends on both antibody specificity and affinity. To characterize the role of antibody affinity, we performed studies in dogs with acute coronary occlusion followed by reperfusion using 3 monoclonal antimyosin antibodies with different affinities. METHODS AND RESULTS: Dogs with experimental reperfused acute myocardial infarction were injected with 2 high-affinity radiolabeled monoclonal antimyosin Fab fragments (R11D10 and 2G42D7), 1 low-affinity antimyosin Fab (3H31E6), and a nonspecific Fab. The left lateral gamma images at 5 H were used to assess the infarct (I) to blood (B) region of interest (ROI) count density ratios by computer planimetry. All infarcts were confirmed by in vivo imaging with 201Tl for perfusion defects as well as by postmortem histochemical staining. The mean I/B ROI (+/-standard deviation [SD]) for R11D10 (1.701+/-0.376) was not significantly different from that of 2G42D7 (1.501+/-0.267, P = NS), but both were significantly greater than that of 3H31E6 Fab (0.85+/-0.12, P = .0001 and .0012, respectively). The I/B ROI of 3H31E6 Fab was similar to that of nonspecific Fab (0.75 to 0.77 range). Radiolabeled R11D10 and 2G42D7 were unequivocally positive by gamma imaging in all infarcts by 5 H. No infarcts were visualized with 3H31E6 or nonspecific Fab. CONCLUSIONS: The low-affinity antibody, despite its specificity for cardiac myosin, cannot be used to image the infarct zone. Therefore immunoscintigraphic diagnosis of irreversible myocardial injury with radiolabeled antimyosin Fab is doubly specific because in vivo visualization required both specificity and high enough affinity of the 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。     

A型肉毒毒素轻链多步纯化及其金属蛋白酶活性研究

目的：利用基因工程方法原核表达N－His标签的A型肉毒毒素轻链（ BoNT－ALC）蛋白,多步纯化获得高纯度蛋白,并对其金属蛋白酶活性进行鉴定。方法以A型肉毒毒素为模板,构建重组表达载体pET22b－ALC,转化BL21（DE3）,诱导蛋白表达,采用镍柱亲和纯化、阴离子交换以及分子筛纯化获取高纯度目的蛋白;通过体外切割特异性底物SNAP－25实验进行酶活性研究。结果与结论获得高纯度的BoNT－ALC蛋白,对金属蛋白酶活性进行了验证。该纯化方法获得的蛋白纯度高、均一性好,且具有很好的酶活性,为后续研究奠定了基础。     

Use of I-131 labeled, murine Fab against a high molecular weight antigen of human melanoma: preliminary experience

High molecular weight antigen (HMWA) is a tumor-associated proteoglycan of human malignant melanoma. I-131 labeled Fab fragments of these specific antibodies were used for preliminary feasibility studies for radioimmunodetection and therapy of human subjects who had inoperable metastatic melanoma. Ten patients received tracer doses of 5-13 mCi (185-481 MBq) of I-131 (anti-HMWA) Fab. All patients (8/8) who had melanoma lesions greater than 1 cm by correlative diagnostic methods had one or more lesions that had localization to tumor of the radiolabeled Fab. In all, 17 of 23 (74%) documented metastases were seen. There were no false positives in this series. Two patients who had avid uptake received potentially radiotherapeutic doses of 142 mCi (5,254 MBq) (one patient) and 181 mCi (6,697 MBq) and 193 (7,141 MBq) (total: 374 mCi or 13,838 MBq) (one patient). For both of these patients, whole body imaging studies showed that the localization of the high dose I-131 Fab was predominantly in tumor. The patient who received the larger dose showed a greater than 50% reduction in the size of pelvic and pericaval nodes, with stabilization of disease at the smaller nodal size for a period of three months. On whole body images, the anti-Fab HMWA appears to be more tumor selective than Fab preparations that target the p97 antigen for melanoma, and there is less uptake in liver.     

Effect of Zr:Mo ratio on 99mTc generator performance based on zirconium molybdate gels.

Zirconium molybdate gels have shown to be viable alternatives for preparation of 99mTc generators using 99Mo produced by neutron activation. The aim of this work was to investigate the effect of the Zr:Mo molar ratio on the gel chemical structure and correlate it with the elution efficiency. A series of gels were prepared at Zr:Mo molar ratios from 0.5:1 to 2.3:1 and characterized by TGA, IR, XRD and UV. It was found that the variation of Zr:Mo ratio produces different polymolybdate arrangements on the octahedral units around to the zirconia which is mainly influenced by the water content. When the matrix molybdenum concentration was increased a lesser amount of water was found and the elution efficiencies were increased. However high elution efficiencies produce higher 99Mo breakthrough values. The gel formulation appears thus to be a compromise between the elution efficiency and the molybdenum breakthrough. The chemical-physical properties of these gels are presented and discussed.     

Labeling of monoclonal antibody conjugates with 90Y

An anti-carcinoembryonic antigen (CEA) antibody, NP-4, was labeled with ⁹⁰Y using p-isothiocyanatobenzyl DTPA (SCN-Bz-DTPA) and its derivatives 1-(p-isothiocyanotobenzyl)-3-methyl-DTPA (1B3M),0 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), 1-(2)-methyl-4-isothiocyanotobenzyl-DTPA (MX-DTPA) as the chelating agents. The ⁹⁰Y conjugates were purified from unbound ⁹⁰Y by two different methods, HPLC or acrylamide size exclusion gel chromatography, in order to evaluate the best purification method. Labeling efficiency, reaction kinetics and immunoreactivity were compared to the same antibodies labeled with [¹¹¹In]citrate. Labeling efficiency, as determined by either HPLC or ITLC (instant thin layer chromatography), was consistently higher by ITLC than HPLC for ⁹⁰Y-labeled MAb, but equal for ¹¹¹In-labeled MAbs. Discrepancies between the 2 methods were linked to impurities in the ⁹⁰Y that remained at the origin of ITLC plates. After purification by acrylamide gel filtration, recovery was 50–60% of loaded ⁹⁰Y activity, but was more than 87% for the ¹¹¹In compounds. Using HPLC, the recovery measured 85% for ⁹⁰Y-labeled MAb and more than 93% for ¹¹¹In-labeled conjugates. Immunoreactivity of the [⁹⁰Y]MAb was comparable to the ¹¹¹In-labeled conjugates. These studies indicate that HPLC purification of the [⁹⁰Y]MAbs improves recovery of activity, and suggests that impurities found in the ⁹⁰Y and metal-binding properties of acrylamide may have contributed to the poor recoveries from acrylamide gels.