抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。     

人突触小体相关蛋白25单克隆抗体的制备及初步鉴定

目的:制备抗人突触小体相关蛋白25(SNAP25)的鼠源单克隆抗体。方法:利用大肠杆菌表达SNAP25蛋白,纯化后免疫BALB/c小鼠制备杂交瘤细胞,筛选针对SNAP25的阳性杂交瘤细胞株,鉴定抗体亚型;用杂交瘤细胞株制备腹水单抗,纯化后利用SDS-PAGE检测抗体纯度。结果:表达并纯化得到纯度大于90%的SNAP25蛋白,免疫小鼠后经2轮筛选得到12株阳性杂交瘤细胞株,其中抗体重链包括IgG1、IgG2型,轻链大部分为κ链;选择具有相对较高抗原结合活性的14号杂交瘤细胞株制备腹水,纯化后得到纯度大于90%的抗体。结论:获得1株高纯度的针对SNAP25的鼠源单克隆抗体,为肉毒毒素的检测奠定了基础。     

6C6鼠-人嵌合抗体的纯化及鉴定

采用基因工程技术 ,将小鼠 6C6单克隆抗体可变区基因与人抗体恒定区基因连接 ,构建了鼠 人 6C6嵌合抗体基因 ,并在CHO细胞中高效表达 .利用ProteinA亲和层析柱从细胞培养上清中分离纯化 6C6嵌合抗体 ,得到电泳纯度大于 98%的 6C6嵌合抗体 ,其重链 (5 5kD)和轻链 (2 4kD)符合IgG相对分子质量的理论值 .Western印迹、细胞免疫荧光和免疫组织化学实验结果均呈阳性 .表明6C6嵌合抗体可识别人乳腺癌细胞表面上的肿瘤相关抗原 ,保持了 6C6单克隆抗体的特性 ,为后续的研究工作奠定了基础     

芜菁花叶病毒单克隆抗体的制备及检测应用

先以芜菁花叶病毒（TuMV）免疫BAL B/C小鼠,然后取其脾细胞使之与SP2/0鼠骨髓瘤细胞融合,经筛选、克隆,获得4株能稳定传代并分泌抗TuMV单克隆抗体（Mab）的杂交瘤细胞,并以之制备腹水单抗。4株单克隆抗体腹水ELISA效价在10-5～10-6之间,仅对TuMV起特异性反应。Western blot分析表明,4株单抗都能与TuMV 34kD的外壳蛋白亚基起特异反应。利用TuMV的多抗兔血清和单抗腹水建立了三抗体夹心ELISA检测TuMV的方法,检测病叶的灵敏度为1∶5120倍,检测提纯TuMV病毒绝对量为21.9 ng。利用三抗体夹心ELISA测定出7种作物上有TuMV侵染。      

SARS-CoV单克隆抗体的制备及抗原表位的初步鉴定

参照已发表的SARS冠状病毒BJ01株基因序列 ,利用计算机软件预测并选取该病毒S、M、N三种主要结构蛋白部分抗原性优势区域 ,以编码Gly-Pro-Gly序列相连接合成两段嵌合基因A和B。并分别克隆于pGEX -6p- 1载体上用IPTG进行诱导表达 ,以纯化的嵌合蛋白A和B为抗原 ,分别免疫BALB c小鼠制备单克隆抗体。利用单克隆抗体亚型检测试剂盒和SARS CoV商品化ELISA检测试剂盒对其进行亚型和特异性鉴定。结果表明融合表达两段嵌合基因产物 ,其大小分别为 34kD和35kD ,Westernblot分析证实两种表达产物都能被SARS病人康复期血清所识别。获得了 6株能稳定分泌特异性抗体的阳性细胞克隆株。亚型鉴定结果除D3C5为IgG2a外其他单抗均为IgG1,而且所有单抗的轻链均为κ链。特异性鉴定发现除D3D1外 ,其余的 5株单抗均能与SARS CoV商品化ELISA检测试剂盒发生特异性反应。将D3D1与灭活后经超声波裂解的SARS CoV进行Westernblot分析 ,发现它能特异性识别 180kD的蛋白带。分别融合表达了 6个S蛋白的寡肽 (S1- S6 ) ,并对筛选出的单克隆…     

蚕豆萎蔫病毒单克隆抗体制备及检测应用

:用蚕豆萎蔫病毒(BBWV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得6株能稳定传代并分泌抗BBWV单克隆抗体(Mab)的杂交瘤细胞株,单抗腹水ELISA滴度为1:320000～1:640000,各单抗抗体类型均为IgG1。6株单抗与BBWV不同分离物均有反应,而与其它植物病毒无交叉反应。经Westernblot印迹分析表明,此6株单克隆抗体均是针对BBWV447kD的外壳蛋白大亚基的特异性抗体。这是国内外首次报道获得BBWV单克隆抗体     

1组曲霉单克隆抗体的制备与鉴定

目的制备并鉴定一组抗曲霉不同抗原的单克隆抗体。方法采用烟曲霉细胞壁抗原成分、分泌抗原和灭活分生孢子,分别免疫BALB／c小鼠,制备单克隆抗体,免疫荧光法鉴定单克隆抗体与曲霉属和念珠菌属抗原的交叉反应。结果获得29株稳定分泌抗曲霉单抗的杂交瘤细胞株,其中用烟曲霉细胞壁抗原成分免疫获得11株,用分泌抗原免疫获得13株,用孢子免疫获得5株;Ig亚类鉴定,11个克隆株为IgG1亚类,3个克隆株为IgG3,15个克隆株为IgM。免疫荧光法鉴定29株单抗特异性识别烟曲霉细胞壁抗原,与其他曲霉抗原有交叉反应。结论29株单克隆抗体,对于建立侵袭性曲霉感染早期诊断方法、筛选曲霉保护性抗体以及研究抗体保护机制奠定了实验基础。     

牛IFN-γ原核表达、单克隆抗体制备及其ELISA检测方法的建立

实验旨在建立牛重组IFN-γ(BovIFN-γ)的ELISA检测技术,为牛传染病的免疫学诊断提供新方法。PHA刺激体外培养的奶牛外周血白细胞,从培养细胞中提取总RNA,经过RT-PCR扩增出BovIFN-γ基因cDNA,进一步克隆至pET28a,转化大肠杆菌,经IPTG诱导,表达出预期大小(18kD左右)组氨酸标记蛋白,经鉴定为BovIFN-γ;以纯化的重组BovIFN-γ为免疫原,应用淋巴细胞杂交瘤技术,获得4株能稳定分泌抗BovIFN-γ单克隆抗体的细胞株,分别命名为A7、A10、G6与G10。免疫球蛋白亚类鉴定证明杂交瘤细胞所分泌的抗体均为IgG1,腹水效价在1∶210×100~1∶211×100之间。Western-blot分析显示,4株单抗均能特异性结合重组BovIFN-γ。ELISA试验表明,4株单抗只与融合蛋白BovIFN-γ反应,而不与非相关性蛋白Ag85B、ESAT-6-CFP-10、GM-CSF等发生反应。选取A10细胞株分泌的单克隆抗体、纯化的多克隆抗体及辣根过氧化物酶(HRP)标记的羊抗兔IgG,建立了检测BovIFN-γ的双抗体夹心ELISA方法。实验结果表明,此方法检测敏感性达到2ng/mL,特异性良好,为进一步建立灵敏、特异的病原感染诊断方法奠定了基础。     

玉米内州萎蔫病菌免疫学检测方法的建立

以玉米内州萎蔫病菌单抗(4H4和4G12)为基础,纯化抗体后,进行亚类鉴定、效价及特异性测定。比较间接ELISA和双单抗夹心ELISA(DAS-ELISA)的检测灵敏度,并应用于玉米种子中萎蔫病菌的检测。结果表明,两株单克隆抗体(0.4g/L)效价均可达1:256000,亚类鉴定结果分别为IgG2a和IgG2b,轻链均为K链。与供试的16株非目标细菌均无交叉反应。DAS-ELISA对萎蔫病菌种子悬浮液的检测灵敏度为1.0×109CFU/L,在此基础上建立了灵敏、特异的玉米内州萎蔫病菌双单抗DAS-ELISA检测方法。     

人源抗狂犬病毒中和性全抗体在昆明细胞中的表达

将来源于噬菌体抗体库的人源狂犬病毒糖蛋白特性异特单抗G10Fab基因的,克隆入杆状病毒人源IgG抗体表达载体,通过转染将重组质粒导入昆虫细胞,以全抗体的形式表达了一株人源抗狂犬病毒基因工程抗体R10。用亲和层析的方法纯化了表达产物,经过一株鼠源糖蛋白特异性单抗竞争证实,该单克隆抗体特异性识别狂犬病毒糖蛋白,亲和力约为10^-9M.体外中和实验证明,该单抗对狂犬病毒aG株具有体外中和活性。     

Mitochondrial genome of Silurus asotus (Teleostei: Siluriformes)

The complete mitogenome sequence of the Amur catfish Silurus asotus was determined using long PCRs. The genome was 16,528 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region; the gene composition and order of which was similar to most other vertebrates. The overall base composition of the heavy strand is 30.5% A, 25.8% T, 28.0% C, and 15.8% G, with an AT content of 56.3%. The mtDNA sequence of S. asotus shared 93.6% and 90.6% sequence identity with that of Silurus meridionalis and Silurus glanis. This mitogenome sequence data would play an important role in silurid catfish phylogenetics and siluriform catfish systematics in general.     

兰州鲇与鲇消化系统的形态学及组织学比较研究

为探究黄河濒危鱼类兰州鲇（Silurus lanzhouensis）消化系统的形态学和组织学结构特点,以鲇（Silurus asotus）为对照,对兰州鲇消化系统形态学和组织学进行了深入研究。结果表明:（1）兰州鲇与鲇的消化道和消化腺形态相似,具有肉食性鱼类的特征。兰州鲇消化道较短,有发达的“U”型胃,胃内皱褶明显,无幽门盲囊,肠道短且粗,可分为前肠、中肠和后肠三部分,前肠粗大,后肠较细。两种鲇属鱼类都有独立致密的肝脏和胰脏。（2）兰州鲇的比肠长显著大于鲇（P < 0.05）,比胃重、比肝胰脏重显著低于鲇（P < 0.05）,但二者的比肠重无显著性差异（P>0.05）。（3）兰州鲇胃的皱襞幅度小于鲇,且环肌层比兰州鲇薄。兰州鲇与鲇前肠的肠黏膜均形成了大量皱襞,肠黏膜、褶皱粗大,但鲇的褶皱分支较细密。兰州鲇与鲇的后肠与前肠相比,肠腔变小,褶皱数量明显减少,高度降低。黏膜层分布有杯状细胞和柱状细胞。兰州鲇与鲇的肝脏肝小叶间缺少结缔组织,分界不明显,而兰州鲇肝细胞的密度大于鲇。综上所述,兰州鲇与鲇的消化系统相似,均符合肉食性鱼类消化系统特征,结合消化生理等研究结果,表明兰州鲇的消化能力弱于鲇,这可能是在自然情况下兰州鲇的分布区域及适应性不及鲇的原因之一。     

南方鲇血型的初步鉴定

本研究旨在通过观察南方鲇血清与其红细胞的交叉反应以鉴定南方鲇的血型.实验结果表明:南方鲇的血清与同种其他个体的红细胞进行交叉反应时均未出现凝集现象,这表明南方鲇可能不存在血型或南方鲇具备血型但血清中相应的凝集素含量不足.以南方鲇的红细胞为抗原免疫日本种大耳白兔制备的抗血清与南方鲇的红细胞进行交叉反应,出现了不同程度的凝集反应,这表明南方鲇存在血型.据上述两个实验结果可以推断,南方鲇可能存在4种血型,分别命名为NA、NB、NAB和NO型;同时也证实,在鉴定南方鲇血型的研究中,通过制备抗血清与红细胞进行交叉反应的方法更为可靠.     

Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays

Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.     

动物特殊动力作用的研究进展

特殊动力作用（SDA）是指动物摄食过程中的代谢产热增加的现象,自上世纪初,一直受到相关领域专家的关注。近十年来该方面研究十分活跃。我们以南方鲇（Southern catfish／Silurus meridionalis）和鲇鱼（catfish／Silurus asotus）为实验对象,开展了大量相关研究。本文就我们的研究成果结合相关研究进行探讨,并指出了该方面研究将来可能的方向。     

Monoclonal antibodies to snakehead, Channa striata immunoglobulins: detection and quantification of immunoglobulin-positive cells in blood and lymphoid organs

Snakehead Channa striata is an important freshwater food fish in many Southeast Asian countries. Three monoclonal antibodies (C9, C10 and D10) were developed against purified serum immunoglobulins of Channa striata (Cs-Ig) and characterized. C9 and D10 MAbs were specific to heavy chain, while C10 MAb detected only unreduced Cs-Ig in western blotting. In competitive ELISA, C9 and C10 MAbs were specific to C. striata Ig and showed no cross reactivity with serum Ig of other fish species i.e. Channa punctatus, Channa marulius, Clarias batrachus and Labeo rohita. D10 MAb showed reactivity to serum Ig of C. striata and C. marulius. In FACS analysis of gated lymphocytes, the percentage of Ig+ cells detected by C9 MAb was 18.2%, 27.7% and 10.3% in blood, spleen and kidney, respectively (n=3, body weight 500-600 g). However, only a few cells (0.5%) were found to be Ig+ in thymus (n=5). C9 MAb was also successfully employed to demonstrate Ig+ cells in blood smears and formalin fixed sections of spleen and kidney. These findings suggest that the spleen plays an important role in humoral immunity as compared to head kidney. Further, these MAbs can be useful immunological tool in monitoring health status of cultured C. striata.     

南方鲶性腺分化的组织学观察

用芳香化酶抑制剂(Fadrozole)、雌激素受体拮抗剂(Tamoxifen)对人工孵化的南方鲶(Silurus meridionalis)幼鱼进行雄性化诱导处理(口服),获得雄鱼。对孵化后第5—130d的南方鲶幼鱼性腺进行组织学观察,结果表明,在实验条件下,南方鲶性腺分化发生在孵化后7d左右,雌雄性分化过程差异明显。雌鱼卵巢腔在孵化后12d左右形成,生殖细胞在孵化后35d左右快速增殖,成熟分裂最早发生在孵化后55d左右;雄鱼生殖细胞在孵化后130d左右快速增殖,成熟分裂最早发生在孵化后130d左右。雌性性腺分化早于雄性。     

Nutritional homeostasis in carnivorous southern catfish (Silurus meridionalis): is there a mechanism for increased energy expenditure during carbohydrate overfeeding?

In previous growth experiments with carnivorous southern catfish (Silurus meridionalis), the non-fecal energy lose was positively related to dietary carbohydrate level. To test whether metabolic energy expenditure accounts for such energy loss, an experiment was performed with southern catfish juveniles (33.2-71.9 g) to study the effect of dietary carbohydrate level on fasting metabolic rate and specific dynamic action (SDA) at 27.5 degrees C. The fasting metabolic rate in this catfish was increased with dietary carbohydrate level, and the specific dynamic action (SDA) coefficient (energy expended on SDA as percent of assimilated energy) was not affected by dietary carbohydrate level. The results suggest that in southern catfish, carbohydrate overfeeding increases metabolic rate to oxidize unwanted assimilated carbohydrate. A discussion on the poor capacity of intermediate metabolism for adapting dietary carbohydrate in carnivorous fish and its possible relationship with facultative component of SDA was also documented in this paper.     

Pattern of energy allocation in the southern catfish (Silurus meridionalis)

Energy budgets were compiled for the southern catfish ( Silurus meridionalis ) under laboratory conditions at different ration size, body weights and temperatures. Though the food energy allocated among the components of the energy budget varied with changing conditions, for the fish fed maximum rations the pattern of energy allocation was independent of body weight and temperature. At the maximum ration about 60% of assimilated energy was deposited in growth and 40% spent in metabolism, indicating a higher proportion allocated to growth and a lower proportion to metabolism than is generally found in fish. This pattern of energy allocation is an energetic strategy adapted to the piscivorous lifestyle of this species.