单链抗体的研究进展及其应用前景

随着分子生物学的进一步发展，抗体技术也逐步由细胞工程抗体(单克隆抗体)向基因工程抗体演替，特别是单链抗体在理论和应用方面的研究尤为令人关注。本文对单链抗体的特点及单价单链抗体、单链抗体多聚体、双特异性单链抗体等方面的研究进展进行回顾，并就单链抗体的应用前景进行了简单地阐述。     

单链抗体的研究进展及其应用

随着分子生物学的发展,单链抗体作为一种小分子基因工程抗体,在理论和应用方面的研究引起了广泛的关注,其在显像定位诊断、靶向治疗和参与细胞免疫等方面展示出了可观的应用前景。对单链抗体的结构及特点、制备技术及其应用等进行了综述,以期能够为单链抗体的进一步深入研究和应用提供理论基础。     

抗氯霉素单链抗体基因工程菌表达条件优化

本实验通过绘制工程菌的生长曲线,确定其生长规律,从诱导物、诱导时机、诱导时间三个主要因素考察其对单链抗体表达的影响,旨在优化抗氯霉素单链抗体基因工程菌的表达条件结果表明:选择IPTG作为诱导物,在工程菌对数生长中期3 h开始诱导,诱导表达4 h结束,单链抗体的表达量可占菌体总蛋白的45%,为抗氯霉素单链抗体的进一步研究和生产应用打下基础。     

单链抗体及其在肿瘤诊断和治疗中应用研究进展

单链抗体(ScFv)是将抗体重链可变区和轻链可变区通过一段连接肽连接而成的重组蛋白,也是目前报道最多的基因工程抗体。ScFv具有分子质量小,免疫原性低,无Fc端,不易与具有Fc受体的靶细胞结合,对肿瘤组织的穿透力强,可以与其他效应分子连接成抗肿瘤融合蛋白等特点,是保持抗体亲和性和特异性的最小功能性抗体片段,广泛应用于医学和生物技术研究领域。文章介绍了ScFv的特点、基因来源、构建、表达及其在肿瘤的诊断和治疗中的应用研究进展。     

氨肽酶N鼠源单链抗体T7噬菌体展示文库的构建

氨肽酶N(pAPN)是一种常见的猪传染性胃肠炎病毒(TGEV)和猪流行性腹泻病毒(PEDV)的细胞受体。为研究抗pAPN的单链抗体(scFv),试验设计一系列简并引物,通过RT-PCR方法从免疫了天然pAPN的BALB/c小鼠脾中克隆了免疫球蛋白轻链可变区(VL)和重链可变区(VH)的编码基因。通过重叠延伸PCR(SOE-PCR)用12个氨基酸的连接肽将VL和VH的扩增产物随机连接,从而获得单链抗体基因。将整个单链抗体基因与T7Select10-3b载体连接,通过体外包装获得了针对pAPN的鼠源ScFv噬菌体文库。pAPN单链抗体初级文库包含2.0×107个重组噬菌体克隆,扩增后的文库滴度达到3.6×109 PFU/mL。Bst NⅠ酶切分析和DNA测序结果显示,28个pAPN抗体初级文库的噬菌体克隆多样性良好。以pAPN C亚基重组蛋白为包被抗原,用噬菌体ELISA进一步验证抗pAPN单链抗体库的活性。本研究主要介绍了采用T7噬菌体展示系统构建猪冠状病毒TGEV和PEDV通用受体pAPN的鼠源单链抗体库及其鉴定。     

基因工程单链抗体的研究进展及临床应用

基因工程抗体在医学生物学研究、疾病的诊断治疗及其他方面有着广泛应用。近年来国内外学者对基因工程抗体进行了广泛的研究,涉及到抗体基因的获取、构建、表达及最后的应用。尤其是单链抗体（single chain antibody,SCA ＆ single chain Fv,ScFv）,因其具有抗原亲活性、分子量小、穿透力强、体内循环半衰期短及排除了Fc段所致的免疫复合物反应等优点,而成为基因工程抗体研究领域中的热点课题。特别是将抗体进行人源化改造、与免疫毒素连接形成融合蛋白的研究已取得突破性进展,目前已有多个人源性ScFv抗体获美国FDA的批准。笔者认为,如果能进一步提高其稳定性及表达产量,开拓免疫毒素及其它新型免疫融合蛋白的研究范围,制备出性能稳定、特异性强、亲和力高、效能多样的基因工程抗体,就可在更大范围取得更大的成绩。     

大容量天然鼠源核糖体展示单链抗体文库的构建与鉴定

为了构建大容量、天然鼠源核糖体展示(ribosome display,RD)单链抗体(single-chain fragment variant,scFv)文库。通过提取SPF级小鼠脾脏总RNA,反转录为cDNA,利用引物分别扩增鼠抗体的VH、VL和Cκ基因,采用重叠延伸PCR(SOE-PCR)方法,通过Linker((Gly4Ser)3)相互拼接,构建单链抗体scFv基因文库和核糖体展示基因文库;经连接转化,对所构建基因文库进行菌落PCR鉴定和序列测定分析。所构建核糖体展示基因文库库容量约为5.6×1013,基因片段全长大小约为1 100bp,基因文库序列具有较好的完整性和多样性。本试验成功构建了一个大容量、天然鼠源核糖体展示单链抗体文库,为进一步筛选单链抗体奠定基础。     

堆型艾美耳球虫特异性单抗轻重链可变区基因的制备

单链抗体是一种新型的基因工程抗体[21]由抗体的重链可变区（VH）和轻链可变区（VL）通过一段短肽（Linker）连接而成,其分子质量小,结构简单,较亲本抗体免疫原性低,但却较好地保持了亲本抗体的抗原亲和活性.     

抗产气荚膜梭菌ε毒素Phage-ScFv库的构建、筛选与鉴定

利用本实验室已诱导并纯化后的重组毒素蛋白制成类毒素免疫BALB/c小鼠,提取小鼠脾脏mRNA。通过RT-PCR和SOE-PCR获得由抗体重链可变区(VH)-Linker-轻链可变区(VL)构成的750bp左右单链抗体(ScFv)基因片段。随后利用噬菌体展示技术成功构建了抗产气荚膜梭菌ε毒素噬菌体单链抗体(Phage-ScFv)库。并通过"吸附-洗脱-扩增",5轮高效的淘洗筛选系统,最终筛选出特异性强的噬菌体抗体。最后,对强阳性的单链抗体侵染E.coli HB2151进行表达,SDS-PAGE结果显示其相对分子质量为30 000左右,与预期结果一致,并且能以分泌形式表达。本试验为抗产气荚膜梭菌ε毒素中毒治疗及其分子致病机制的研究奠定了基础。     

抗诺氟沙星噬菌体单链抗体库的构建与筛选

应用噬菌体展示和重组抗体技术制备抗诺氟沙星(NFLX)单链抗体库,筛选获得抗NFLX高特异性、高亲和力的单链抗体scFv。将NFLX与BSA化学偶联获得的免疫源,免疫Balb/C小鼠,提取脾细胞RNA,反转录获得cDNA。以针对鼠源重链可变区(VH)及轻链可变区(VL)基因的特异性引物,扩增获得VH基因和VL基因。通过SOE-PCR法将VH基因和VL基因通过柔性多肽Linker((Gly4Ser)3)拼接成VH-Linker-VL片段,酶切(sfiⅠ+NotⅠ)处理后,插入噬粒载体pCANTAB5E,并转化宿主菌TG1,通过辅助噬菌体M13K07拯救,构建抗NFLX噬菌体单链抗体库。以包被抗原NFLX-OVA包被96孔板,亲和富集法,经三轮淘筛,间接Elisa初步检测重组scFv的特异性抗原结合活性。成功构建噬菌体单链抗体库,获得鼠源抗NFLX特异性的scFv,噬菌体中scFv插入率为90%,获得库容约为1.3×106的抗NFLX噬菌体单链抗体库,筛选得到5株抗NFLX的scFv。为运用噬菌体展示抗体技术制备特异性抗药物单抗及进一步建立药残检测新技术奠定了基础。     

Feline intracranial neoplasia: retrospective review of 160 cases (1985-2001)

The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.     

The Prevalence and Intensity of Helminth and Coccidial Infections in Dairy Cattle in Central Kenya

A survey of gastrointestinal parasite infections of young (<6 months old), immature (6–12 months old) and adult (>12 months old) dairy cattle on 16 farms in Kiambu District, Kenya was conducted during a dry season (September 1991 to January 1992) and during a wet season (March to July 1992). The survey was based on monthly coproparasitological examination of cohorts and worm counts in tracer calves. The effects of age, sex, farm and season on the prevalence and intensity of helminth and coccidial infections were determined. Faecal egg and oocyst counts revealed that the overall prevalences were: strongyles (including trichostrongyles) (85.5%), liver flukes (Fasciola gigantica) (34.0%), coccidia (30.9%) and tapeworms (9.6%). Eight species of the protozoan Eimeria were identified, the most prevalent species being E. bovis and E. zuernii. The most prevalent nematode genera were Haemonchus, Cooperia, Oesophagostomum and Trichostrongylus. Season, farm and age of the animals had a significant (p<0.05) influence on the intensity of infection with strongyles, liver flukes and coccidia, whereas the sex of the animals had no significant (p>0.05) effect on the prevalence or intensity of infections. A higher intensity of infection with strongyles and coccidia was found in the wet season than in the dry season (p<0.05). The age-specific intensity was in the following order: for strongyles, immature animals of 6–12 months of age had the highest egg counts, followed by young calves and adults. Calves had significantly (p<0.05) higher oocyst counts than immatures or adults. Liver fluke egg counts did not differ significant (p>0.05) between immatures and adult cattle.     

Hypothalamic dopamine is required for salsolinol‐induced prolactin secretion in goats

The aim of the present study was to clarify the relationship between hypothalamic dopamine (DA) and salsolinol (SAL) for the secretion of prolactin (PRL) in goats. SAL or thyrotropin‐releasing hormone (TRH) was intravenously injected into female goats treated with or without the D₂ DA receptor antagonist haloperidol (Hal), which crosses the blood‐brain barrier, and the PRL‐releasing response to SAL was compared with that to TRH. PRL‐releasing responses to SAL, Hal, and Hal plus SAL were also examined after a pretreatment to augment central DA using carbidopa (Carbi) and L‐dopa. The PRL‐releasing response to Hal alone was greater than that to SAL or TRH alone. The PRL‐releasing response to Hal plus SAL was similar to that of Hal alone. In contrast, the PRL‐releasing response to Hal plus TRH was greater than that to TRH or Hal alone. The treatment with Carbi plus L‐dopa inhibited SAL‐ and Hal‐induced PRL secretion. The inhibition of the PRL‐releasing response to SAL disappeared when SAL was injected with Hal. These results indicate that the mechanisms underlying the SAL‐induced PRL response differ from those of TRH, and suggest that hypothalamic DA and its synthesis is associated in part with SAL‐induced PRL secretion in goats.     

Laboratory diagnosis of respiratory diseases of cattle

The authors describe the procedure of laboratory diagnosis for bovine respiratory diseases: direct diagnosis by isolation and for identification of bacteria or viruses and indirect diagnosis by serological methods. They specify the restraints and limits of this diagnosis and the significance results which are obtained and connected with knowledge of anamnestic information.     

The effect of peri-parturient anthelmintic treatment on the productivity of dairy cattle in subtropical western India

Trials with fenbendazole (Panacur, Hoechst India Ltd) were carried out on two commercial farms in subtropical western India to study the response and economics of nematode treatment in adult dairy cows. Milk yield, lactation length, time to first oestrus and worm egg output were monitored in treated and control groups. Treatment reduced the egg count considerably. Treated cows produced 142 litres more milk over 100 days (p<0.05), with extension of lactation length and advancement of time to first oestrus. The economic gain in terms of milk yield far outweighed the cost of anthelmintic used.     

An Evaluation of the Acceptability as Forage of Some Nutritive and Antinutritive Components and of the Dry Matter Degradation Profiles of Five Species of Ficus

The suitability of five species of Ficus, F. mucoso, F. thonningii, F. polita, F. religiosa and F. benjamina, for feeding ruminant livestock was studied. The nutritive and antinutritive components were determined and the acceptability of the forages to livestock was assessed using a cafeteria method in 8 adult small ruminants. Also, the degradation potential of the dry matter of the five Ficus species was investigated using 3 fistulated sheep. The crude protein (CP) content of the Ficus species varied significantly (p < 0.05), ranging from 130 to 180 g/kg DM. The content of neutral detergent fibre (NDF) also varied significantly (p < 0.05), ranging from 650 to 710 g/kg DM. The organic matter (OM) contents did not differ significantly (p > 0.05), having a mean value of 916 g/kg DM. The concentrations of tannin, saponin, phytic acid and oxalic acid were low. The acceptability of the forage was similar for four of the species but poor for F. polita. The extent of DM degradation varied significant among the Ficus species, the ranking order being F. benjamina > F. thonningii > F. mucoso > F. religiosa > F. polita.     

Nutrient and metal analyses of Chinese herbal products marketed for veterinary use

Many Chinese herbs and herbal mixtures are fed to domestic animals for their reputed medicinal properties. These herbs could contribute to the intake of essential nutrients and toxic metals, but their composition is mostly unknown. The purpose of this study was to measure major nutrient (crude protein, crude fat, carbohydrate, fibre) and mineral (Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo, S, Al, Cd, Ni, Pb) concentrations in samples of fourteen combination formulas labelled for veterinary use and commonly administered to horses and dogs. Three single herbs, Bupleurum chinense, Curcuma zedoaria and Astragalus membranaceus, each obtained from several sources, and Yunnan Baiyao, a proprietary hemostatic mixture, were also analysed. Proximate analyses and some mineral concentrations differed (p < 0.05) among single herbs, and high concentrations of several minerals were detected in some herbal combinations. Those containing the highest concentrations [g/kg dry matter (DM)] of calcium (92.4), iron (2.6) and manganese (0.28) could provide >38%, 142% and 96%, respectively, of recommended allowances in adult dogs, and >13%, 122% and 2%, respectively, of maintenance requirements in horses, at the maximum labelled dose assuming complete availability. Concentrations of cadmium, nickel and lead were below published oral tolerance levels. Aluminium concentrations (median 380, maximum 920 mg/kg DM) were higher than has been previously reported in Chinese herbs. These nutrient analyses suggest that herbal combinations marketed to veterinarians, when fed at the maximal labelled dose, are unlikely to produce clinically relevant changes in the dietary intake of essential nutrients. However, small amounts of non‐essential contaminant minerals are present in some formulas, and further research is necessary to understand the significance of this finding.