A型肉毒毒素单克隆抗体的制备和鉴定

目的:制备并鉴定抗A型肉毒毒素重链C端(BoNT/AHc)鼠源单抗。方法:以BoNT/AHc蛋白为抗原免疫小鼠,利用杂交瘤技术获得鼠源单抗,通过抗体分型、SDS-PAGE、Western印迹及ELISA法分析鉴定。结果:筛选得到3株鼠单抗1B1、1B2、1C6,重链类型均为IgG1,轻链类型均为κ型;3株纯化抗体的纯度达90%以上,均能与抗原BoNT/AHc特异性结合,亲和力常数分别为1.43×10~8、2.31×10~8、1.44×10~9L/mol;叠加ELISA结果显示3株抗体与抗原BoNT/AHc结合位点相同或相近。结论:筛选得到3株能与BoNT/AHc特异性结合的鼠源单抗,为A型肉毒毒素中和抗体的研发,以及快速有效的肉毒毒素检测方法的建立奠定了基础。     

鼠源E型肉毒毒素免疫噬菌体单链抗体库的构建及筛选

目的:构建鼠源E型肉毒毒素(BoNT/E)免疫噬菌体单链抗体库,筛选BoNT/E特异性抗体。方法:从E型肉毒类毒素免疫小鼠的脾细胞中提取总RNA,反转录成cDNA,分别扩增出小鼠重链可变区基因和轻链可变区基因;通过重叠延伸PCR将重链可变区基因和轻链可变区基因组装成scFv基因,重组于噬粒pS100中,电转化大肠杆菌TG_1,合并所有克隆成初级库;随机挑取克隆进行核苷酸序列测定,对初级库序列多样性进行分析;在辅助噬菌体M_(13)K_(07)的拯救下,构建成scFv噬菌体抗体库;用纯化的BoNT/E对鼠源BoNT/E免疫噬菌体单链抗体库进行3轮富集筛选,制备单克隆的噬菌体抗体颗粒进行酶联免疫吸附试验,阳性克隆进行核苷酸序列测定。结果:鼠源BoNT/E免疫噬菌体单链抗体库的库容为7.09×10~7,随机挑取的20个克隆序列各不相同,序列正确率为85%,基本覆盖了IgHV、IgKV、IgLV的优势家族;纯化的BoNT/E作为抗原通过3轮筛选,噬菌体抗体富集了66倍,第3轮筛选后随机挑取90个克隆制备噬菌体抗体颗粒,酶联免疫吸附试验分析有88个呈现阳性反应,序列比对得到了24个不同序列的BoNT/E特异性抗体。结论:构建了库容量达7.09×10~7的鼠源BoNT/E免疫噬菌体单链抗体库,筛选得到了24个不同序列的BoNT/E特异性抗体。     

抗H5N1病毒嵌合IgA抗体基因的构建及其在CHO细胞中的表达

为了表达具有中和活性的抗禽流感H5N1病毒人-鼠嵌合IgA抗体,采用RT-PCR法克隆具有中和活性的抗禽流感H5N1-HA鼠源单克隆抗体的轻重链可变区基因及相应的信号肽编码序列,分别与人免疫球蛋白IgA2重链恒定区、Kappa恒定区基因拼接,构建表达质粒pEF-IGHA9和pEF-IGK9,共转染二氢叶酸还原酶缺陷型CHO(CHO-dhfr-)细胞,用ELISA检测培养上清中嵌合IgA抗体的表达,对纯化的嵌合抗体进行SDS-PAGE、Western blotting印迹分析。结果成功地在CHO细胞中表达了抗禽流感H5N1病毒人-鼠嵌合IgA抗体,为制备抗H5N1重组分泌型IgA预防性抗体制剂奠定了良好的基础。     

F型肉毒毒素单克隆抗体的制备与鉴定

目的:制备F型肉毒毒素(BoNT/F)鼠源单克隆抗体。方法:以BoNT/F重链C端(BoNT/FHc)为抗原免疫BALB/c小鼠,取免疫后尾血效价最高小鼠的脾脏细胞与鼠骨髓瘤细胞Sp2/0在聚乙二醇作用下进行细胞融合,ELISA筛选阳性细胞孔,通过有限稀释法分离能稳定分泌抗体的细胞株。小鼠腹腔注射杂交瘤细胞制备的腹水利用Protein G进行亲合层析纯化,SDS-PAGE检测抗体纯度,非竞争ELISA测定抗体亲和力常数,亚型鉴定试剂盒鉴定抗体亚型,PCR扩增抗体轻重链可变区基因并经Vbase软件分析抗体框架区与互补决定区(CDR)氨基酸序列。结果:经过3轮免疫后,5只小鼠尾血效价均达到1∶16 000,其中2号鼠尾血效价最高;杂交瘤细胞融合率为74.48%,阳性率为11.54%;2轮克隆化后筛选得到7株能稳定分泌抗体的杂交瘤细胞株,选择其中细胞培养上清效价最高的1F4制备抗体;腹水纯化后1F4抗体纯度超过95%,与BoNT/FHc抗原的亲和力常数为1.08×10-8mol/L;亚型鉴定结果表明1F4重链和轻链分别为IgG1型和κ型,抗体可变区氨基酸序列分析显示1F4重链和轻链CDR3序列为TRHGYFPSWFAY和QQHYSTPWT。结论:筛选到一株高亲和力的鼠源单克隆抗体,为BoNT/F检测和中和抗体的研发奠定了基础。     

抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器的制备与验证

目的:构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体并制备和验证抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型。方法:利用PCR法扩增出抗人p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L,然后分别将嵌合抗体重链基因H和嵌合抗体轻链基因L连接到乳腺特异性表达质粒pBC1,从而构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L。分别将抗p185~(erbB2)人鼠嵌合抗体ChAb26乳腺特异表达载体pBC1-H和pBC1-L线性化,然后使用原核显微共注射法获得8只转基因FVB小鼠,通过鼠尾直接PCR鉴定其转基因阳性。通过RT-PCR、荧光定量PCR鉴定转基因小鼠乳腺组织中抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达。使用小鼠乳汁采集器收集其乳汁并通过Western blot和夹心ELISA等实验鉴定抗p185~(erbB2)人鼠嵌合抗体ChAb26是否获得表达。结果:经测序验证,抗p185~(erbB2)人鼠嵌合抗体ChAb26的嵌合重链基因H和嵌合轻链基因L分别与乳腺特异表达质粒pBC1正确正向连接。鼠尾直接PCR结果显示所获8只转基因FVB小鼠均为转基因双阳性小鼠,且抗p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L在它们的后代中稳定遗传,它们的后代中转基因小鼠双阳性率约为30%; RT-PCR和荧光定量PCR的结果显示,转基因双阳性小鼠及其双阳性后代的乳腺组织中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达; Western blot和ELISA等实验结果显示,转基因双阳性小鼠乳汁中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的蛋白质表达,而且抗p185~(erbB2)人鼠嵌合抗体ChAb26与羊抗人κ链抗体和羊抗人Ig G Fc-HRP抗体均能特异性结合。结论:成功构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L和制备了抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型,为今后抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因牛乳腺生物反应器的研究奠定了理论和技术基础。     

鼠源抗B型肉毒毒素Fab抗体库的构建及初步筛选

目的:应用重组噬菌体抗体库技术制备抗B型肉毒毒素Fab抗体。方法:用重组B型肉毒毒素重链C端片段(BoNTB/Hc)免疫BALB/c小鼠,从其脾淋巴细胞扩增免疫球蛋白Fd段和κ链基因,克隆至表达载体pComb3中,并将抗体Fab段表达于噬菌体表面,建立容量为5.96×106cfu的噬菌体抗体库。以BoNTB/Hc为抗原对所建抗体库进行4轮亲和筛选,获得与B型肉毒毒素特异性结合的克隆,并进行序列测定。结果:构建了抗B型肉毒毒素Fab抗体库,筛选出特异性克隆1个。结论:从鼠源噬菌体免疫抗体库中初步获得了特异性抗B型肉毒毒素的Fab抗体。     

一种人源抗狂犬病病毒单克隆抗体的瞬时表达及性质分析

目的瞬时表达人源重组抗狂犬病病毒单克隆抗体,并对抗体性质进行分析。方法 PCR法扩增抗体轻、重链可变区并分别构建真核表达质粒;瞬时转染HEK293 EBNA1细胞;抗体经亲和纯化后,CE-SDS法分析纯化后抗体单体比例,用快速荧光灶抑制试验(RFFIT)分析抗体体外抗狂犬病病毒中和活性,ELISA分析抗体与3aG、CTN、PV及CVS株的结合。结果提取瞬时表达质粒的A260/280 nm比值在2.0～2.1之间纯化后抗体非还原CE-SDS单体纯度为74.4%;还原CE-SDS抗体轻、重链峰合计占比超过95%;抗体RFFIT体外中和活性为293.37 IU/mL,比活达628.21 IU/mg,抗体与3aG、CTN、PV及CVS株交叉反应均为阳性。结论该株抗体具有较高体外抗狂犬病病毒中和活性,与主要疫苗株均有较强结合。     

A型肉毒聚合类毒素的制备及免疫原性研究

通过制备A型肉毒聚合类毒素,研究获得较好的免疫原性和反应原性的方法.通过碳二亚胺法,聚合A型肉毒类毒素,免疫小鼠,用ELISA检测抗体,成功地制备了A型肉毒聚合类毒素,免疫小鼠获得高效价免疫血清,且该免疫血清具有中和活性,研究A型肉毒聚合类毒素抗原的作用机制,获得具有中和活性的保护性抗体,对肉毒毒素中毒的防治具有重要意...     

一种从抗狂犬病病毒噬菌体抗体库中筛选和验证中和抗体的方法

目的建立一种从噬菌体库抗体库中高效筛选和验证抗狂犬病病毒中和抗体的方法。方法①定性分析:从经过灭活的CVS-11筛选噬菌体抗体库中挑取单克隆于96孔板中培养,制备噬菌体抗体,取培养上清进行快速荧光灶抑制试验(rapid fluorescent focus inhibition test, RFFIT),选择可明显抑制病毒感染的克隆测序,获得具有中和活性的抗体可变区序列;②定量分析:挑选有中和活性的克隆,重新制备噬菌体抗体颗粒,纯化后进行RFFIT分析;扩增抗体可变区基因,构建真核瞬时表达质粒。瞬转HEK-293 EBNA1细胞,培养上清经Mabselect SuRe亲和纯化后测定抗体比活。结果定性分析获得的噬菌体抗体颗粒与全分子抗体体外中和活性显著相关;剔除低活性序列后,活性高于0.5 IU/mL的噬菌体抗体颗粒与其全分子抗体体外中和活性之间无显著相关;所有纯化后噬菌体抗体颗粒活性>0.5 IU/mL的序列其全分子抗体体外中和活性均>500 IU/mg。结论构建了一种从抗狂犬病病毒噬菌体库中高效筛选、验证中和抗体的方法。     

重组嵌合抗人CD22四价基因工程抗体在昆虫杆状病毒中的表达

重组嵌合抗人CD22四价基因工程抗体是由一条短肽链将两个鼠源抗CD22mAb的scFv连接起来,再与人IgG1的CH3片段连接所获得重组基因工程抗体(cRFB4-CH3),是目前开发治疗B细胞系淋巴瘤的人源化基因工程抗体。为探讨该重组基因工程抗体高效表达技术,本研究利用DNA重组技术将含有人IgG1的CH3段的抗人CD22四价基因工程抗体基因克隆到含信号肽的重组杆状病毒载体pAcSG2中,构建重组质粒pAcSG2-cRFB4-CH3并转染到Sf9细胞中,构建携带有重组嵌合抗人CD22四价基因工程抗体基因的重组杆状病毒AcNPV-cRFB4-CH3。通过对该重组毒进行PCR和IFA鉴定,证实获得了可以稳定表达抗人CD22四价基因工程抗体的重组杆状病毒。以蚀斑试验进一步纯化病毒,经过3次病毒蚀斑克隆,获得毒价达到4.5×107pfu/mL重组病毒,为治疗白血病药物的开发和应用打下了基础。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。     

Cytoskeleton and mitochondrial morphology and function

It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.     

Vascular volumes and hematology in male and female runners and cyclists

To examine the hypothesis that foot-strike hemolysis alters vascular volumes and selected hematological properties is trained athletes, we have measured total blood volume (TBV), red cell volume (RCV) and plasma volume (PV) in cyclists (n = 21) and runners (n = 17) and compared them to those of untrained controls (n = 20). TBV (ml x kg(-1)) was calculated as the sum of RCV (ml x kg(-1)) and PV (ml x kg(-1)) obtained using 51Cr and 125I-labelled albumin, respectively. Hematological assessment was carried out using a Coulter counter. Peak aerobic power (VO2peak) was measured during progressive exercise to fatigue using both cycle and treadmill ergometry. RCV was 15% higher (P < 0.05) in male cyclists [35.4 (1.0), mean (SE); n = 12] and runners [35.3 (0.98); n = 9] compared to the controls [30.7 (0.92); n = 12]. Similar differences existed between the female cyclists [28.2 (2.1); n = 9] and runners [28.4 (1.0); n = 8] compared to the untrained controls [24.9 (1.4); n = 8]. For the male athletes, PV was between 19% (cyclists) and 28% (runners) higher (P < 0.05) in the trained athletes compared to the untrained controls. The differences in PV between the female groups were not significant. Although the males had a higher (P < 0.05) TBV, RCV and PV than the females, no differences between cyclists and runners were found for either gender. Mean cell volume was not different between the athletic groups. VO2peak (ml x kg(-1) x min(-1)) was higher (P < 0.05) in both male [68.4 (1.5)] and female [54.8 (2.1)] runners when compared to the untrained males [47.1 (1.0)] and females [40.5 (2.1)]. Although differences existed between the genders in VO2peak for both cyclists and runners, no differences were found between the athletic groups within a gender. Since the vascular volumes were not different between cyclists and runners for either the males or females, foot-strike hemolysis would not appear to have an effect on that parameter. The significant correlations (P < 0.05) found between VO2peak and RCV (r = 0.64 and 0.64) and TBV (r = 0.82 and 0.63) for the males and females, respectively, suggests a role for the vascular system in realizing a high aerobic power.     

Microbial and bioconversion production of D-xylitol and its detection and application

D-Xylitol is found in low content as a natural constituent of many fruits and vegetables. It is a five-carbon sugar polyol and has been used as a food additive and sweetening agent to replace sucrose, especially for non-insulin dependent diabetics. It has multiple beneficial health effects, such as the prevention of dental caries, and acute otitis media. In industry, it has been produced by chemical reduction of D-xylose mainly from photosynthetic biomass hydrolysates. As an alternative method of chemical reduction, biosynthesis of D-xylitol has been focused on the metabolically engineered Saccharomyces cerevisiae and Candida strains. In order to detect D-xylitol in the production processes, several detection methods have been established, such as gas chromatography (GC)-based methods, high performance liquid chromatography (HPLC)-based methods, LC-MS methods, and capillary electrophoresis methods (CE). The advantages and disadvantages of these methods are compared in this review.