抗HER2-hTNF-α新型免疫毒素的制备及功能研究

HER2/neu基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子.为了增加过度表达HER2/neu的肿瘤细胞对肿瘤坏死因子(TNF)的敏感性和提高HER2/neu抗体的肿瘤杀伤效应,将抗HER2/neu单链抗体C6.5与人肿瘤坏死因子hTNF-α融合,构建了scFvC6.5-hTNF-α融合蛋白,完成了重组蛋白在大肠杆菌中的表达,产率为400μg/L菌液.经过亲和层析和柱复性,融合蛋白的纯度达95%以上.ELISA试验表明,scFvC6.5-hTNF-α能够特异结合HER2/neu阳性卵巢癌细胞SKOV-3和乳腺癌细胞MCF-7,而不结合HER2/neu阴性的黑色素瘤细胞A375.MTT试验表明,scFvC6.5-hTNF-α能够选择性地杀伤SKOV-3和MCF-7细胞,而不影响A375细胞的生长.这种肿瘤细胞特异性杀伤作用提示该免疫毒素具有肿瘤靶向治疗的潜在应用价值.

内源激素含量的变化与白桦成花关系的研究

采用酶联免疫吸附法(ELISA)测定有花史白桦大树与无花史白桦幼树的4种内源激素脱落酸(ABA)、细胞分裂素(iPA)、生长素(IAA)、赤霉素(GA3)含量的动态变化,研究其与白桦成花的关系。研究结果表明:白桦有花史大树与无花史幼树的4种内源激素含量动态变化明显不同。在雌、雄花芽的生理发端期,有花史大树的IAA含量保持相对稳定,而高含量的ABA和iPA含量的逐渐降低以及GA3含量的逐渐升高可能有利于白桦成花。

噬菌体呈现技术筛选抗黑色素瘤单链抗体

目的 :制备黑色素瘤单抗的单链可变区片段 ,用于肿瘤的诊断或靶向治疗。方法 :从杂交瘤细胞提取总RNA,分别扩增出轻重链可变区基因(variableregionofheavychain,VH和variableregionoflightchain,VLDNA) ,连接形成单链抗体 (singlechainfragmentvariableregion,ScFv)DNA,将ScFv与载体pCANTAB5E的连接产物转化大肠杆菌TG1 ,经M13K07超感染后 ,获得噬菌体抗体ScFvcDNA文库 ,用黑色素瘤细胞LiBr对重组的噬菌体抗体进行3轮吸附 -洗脱 -扩增亲和筛选后 ,随机挑选克隆经ELISA筛选鉴定。结果:VH、VL和ScFv分别约为360、330、750bp,从随机筛检的30个克隆中获得10个高亲和性噬菌体呈现型ScFv单克隆。结论:用噬菌体呈现技术制备的黑色素瘤ScFv ,可为肿瘤的诊断和靶向治疗奠定基础

梭形芽孢杆菌(Bacillusfusiforms)N141发酵产河豚毒素的研究

对分离筛选得到的产河豚毒素(TTX)的梭形芽孢杆菌(Bacillus fusiforms)N141菌株,在10L发酵罐中进行发酵产TTX的试验。发酵产物经提取和精制后,用高效薄层色谱、小鼠生物试验,以及河豚毒素单抗ELISA检测试剂盒,检测出发酵液中含有TTX。研究了菌体生长、pH、溶氧、以及产物TTX的变化规律。结果表明TTX是在菌体生长进入稳定期后才产生的,说明TTX是微生物产生的次级代谢产物。10L罐发酵产TTX的工艺技术,在500L发酵罐进行了验证,重现性良好。这为微生物源河豚毒素的发酵中试提供了科学依据。

鸭瘟病毒gB蛋白N端抗原域的高效表达及其免疫特性

在分析鸭瘟病毒(Duck plague virus,DPV)gB蛋白抗原性的基础上,设计1对引物克隆gB蛋白N端抗原性较好的抗原域编码基因,克隆到表达载体pET32a中,构建了原核表达质粒pET-gB1。将pET-gB1转化到感受态大肠杆菌(Escherichia coli)BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约42.4kD的目的蛋白以包涵体形式表达。Western blot分析发现,表达产物与抗鸭瘟的鼠阳性血清发生特异性反应。将包涵体溶解于8mol/L的尿素中,利用His·Bind试剂盒获得纯化的蛋白,将纯化的蛋白皮下注射免疫小鼠,间接ELISA法测得抗体的效价,MTT法检测免疫小鼠的T淋巴细胞增殖反应能力。结果说明,该融合蛋白能够诱导机体产生较强的体液免疫和细胞免疫。

植物生长调节剂对水曲柳组培苗生长及内源激素的影响

以水曲柳成熟种子为外植体材料,在不同增殖培养基中培养,应用间接酶联免疫法(ELISA)测定脱落酸(ABA)、吲哚乙酸(IAA)和玉米素核苷(ZR)3种内源激素,探讨不同细胞分裂素质量浓度和配比对水曲柳组培苗生长及内源激素的影响。研究结果表明:当年采收的水曲柳种子,消毒、浸泡处理后,播出种胚,可以解除水曲柳种子的休眠。增殖培养基中添加不同细胞分裂素都抑制了组培苗的高生长和生物量,但却可以解除组培苗顶端优势,诱导腋芽生长。与此同时,腋芽发生率与内源激素ABA、IAA和ZR质量分数不存在相关性,而与IAA/ZR比值存在负相关,但相关性并不显著。IAA/ZR比值的减少有可能有利于腋芽的诱导。

虎眼万年青多糖对小鼠免疫功能的调节作用

依据虎眼万年青组分中多糖对小鼠巨噬细胞吞噬功能的影响,筛选并优化出60%醇浓度中性多糖活性组分S3,进而研究其对体液免疫、细胞免疫及细胞因子变化的影响,并从细胞和分子水平探讨其作用的机理.方法:采用溶血素测定方法,测定S3对小鼠脾细胞溶血素抗体的诱导作用;采用3H-TdR渗入法,测定S3对ConA诱导小鼠脾淋巴细胞的增殖作用;采用3H-TdR后标记法,测定S3对NK细胞细胞毒活性的影响;以ELISA法测定S3对小鼠脾细胞IL-2产生的影响;利用流式细胞术,检测S3对T淋巴细胞亚群CD3、CD4、CD8、CD4/CD8阳性细胞百分率的影响;采用RT-PCR方法检测IL-2 mBNA的表达水平.结果:S3高、中剂量组能明显增强小鼠脾细胞溶血素抗体的形成(P<0.05);各剂量组均能明显增强ConA诱导的淋巴细胞增殖能力(P<0.001);各剂量组均能增强NK细胞的细胞毒活性并促进IL-2的产生(P<0.001,P<0.01);高、中剂量组CD8阳性细胞百分率明显降低(P<0.001),各剂量组均能显著地提高CD4/CD8阳性细胞百分率(P<0.001);高剂量组可明显促进脾细胞中细胞因子IL-2的mRNA表达,使表达量增加(P<0.05).结论:S3具有较强的增强机体多种免疫功能的作用,可利用开发为一种免疫增强药物.

时间分辨激光荧光光谱技术在免疫分析中的应用

本研究以时间分辨激发荧光光谱分析技术为基础,进行稀土离子标记的激光激发的时间分辨荧光免疫分析(TRFIA)研究.实验以自行合成的二乙三胺五醋酸酐(DTPAA)为双功能螯合剂.用Eu3+标记兔抗人(RAH)IgG抗体,依据解离增强原理(DELFIA),研究了Eu3+-β萘甲酰三氟丙酮(β-NTA)的荧光分辨体系,测定了荧光光谱和荧光寿命,建立了铕离子分析检出方法,其工作曲线范围为1×10-7～1×10-11 g·mL-1,检测限为1×10-13 g·mL-1,相对标准偏差为6.4%.结合TRFIA方法学研究,进行了人血清丙型肝炎病毒抗体(Anti-HCV)检测.并同酶联免疫法(ELISA)对比.取得TRFIA法阳性检测率明显高于ELISA法的结果.

Effect of lanthanum on expression of recombinant allophycocyanin gene in Pichia pastotis

The enhancing effect of lanthanum on gene expression of recombinant allophycocyanin (rAPC), a potential antitumor medicine, in Pichia pastoris was studied. PCR and sequence analysis were used in order to prove whether the APC gene had integrated into the yeast genome. The expression level of the recombinant allophycocyanin (rAPC) in BMMY medium containing LaCl3 was detected by ELISA method. The recombinant allophycocyanin was determined by Western blot. The results show that the recombinant Pichia pastoris chromosome contained allophycocyanin gene. Expression efficiency of rAPC gene in Pichia pastoris was promoted by proper LaCl3 concentration like 2, 5, 10 mmol (.) L-1, among which 5 mmol (.) L-1 was the most effective. The highest expression yield of rAPC in the BMMY medium containing 5 mmol (.) L-1 LaCl3 was 4.4 mg (.) L-1 at 48 h, that was increased by 110% compared with 2.1 mg (.) L-1 of control, in the meantime, the optimum culture time is shortened from 72 to 48 h. The result of western blot analysis indicates that the rAPC consisted of two kinds of subunits with molecular weight of 19 and 21 kDa respectively.

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5 similar to 6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro-particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 mug/mg soluble proteins on average and the highest value was 2.497 mug/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bio-reactor producing high value materials such as edible HBV vaccine was discussed as well.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2-3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.

织纹螺（Nassarius spp.）体内河豚毒素来源的研究

近几十年来，国内沿海地区频繁发生食用织纹螺中毒事件，并导致数十人死亡，这一问题得到了政府相关部门的高度重视。但是，由于织纹螺毒性变化很大，毒素来源不清楚，因此很难预测食用织纹螺中毒事件的发生，这在很大程度上限制了对食用织纹螺中毒事件的有效监测和管理。目前，对于中国沿海有毒织纹螺体内河豚毒素（tetrodotoxin, TTX）的来源还未见过系统研究。本文选取中国沿海常见的半褶织纹螺（Nassarius semiplicatus）、纵肋织纹螺（N. variciferus）和拟半褶织纹螺（N. semiplicatoides sp. nov.）作为实验对象，从毒素的微生物来源与食物链来源这两个角度分别展开研究，以探讨织纹螺体内 TTX 的可能来源，为提出相应的预防管理措施提供科学依据。 首先，我们先后从曾发生过中毒事件的江苏盐城和连云港采集了织纹螺样品，通过小鼠生物测试法和液-质联用分析技术（LC-MS），对织纹螺的毒性和毒素组成进行了测试和分析，分离培养了织纹螺体内及其生活环境中的细菌，应用河豚毒素单克隆抗体酶联免疫检测方法（ELISA）对细菌的产毒情况进行了测试，并通过 16S 核糖体（rRNA）部分基因序列测定对细菌种类进行了初步的分析。研究发现，采自江苏盐城和连云港的半褶织纹螺的毒性分别约为 2 MU/g 和 200 MU/g 组织，体内的毒素成分是河豚毒素及其同系物。从盐城的半褶织纹螺及其生活环境分离的菌株中随机挑出 14 个菌株中，9 个菌株河豚毒素检测结果呈现阳性。从连云港高毒性半褶织纹螺消化腺中分离到的 45 个菌株中，阳性菌株有 21 个。但是，有毒菌株毒素含量较低，毒素含量范围是 15-184ng/g。通过 16S rDNA 部分序列的测序结果发现，大部分有毒菌株与弧菌属（Vibrio）的细菌在遗传序列信息上比较相近。其余有毒菌株分别与希瓦氏菌属（Shewanella）、海单胞菌属（Marinomonas）、黄杆菌属（Tenacibaculum）、动性菌属（Planococcus）、发光杆菌属 (Photobacterium)和气单胞菌属（Aeromonas）的遗传序列比较相近。其中与海单胞菌属、动性菌属和发光杆菌属亲缘关系较近的产毒细菌是首次报道。这一研究表明织纹螺体内及其生活环境中的存在产河豚毒素的细菌，但由于产毒素的量较低，因此可能在织纹螺体内河豚毒素的产生和累积过程并不发挥主要作用。 织纹螺作为一类腐食性的海洋动物，也有可能通过进食含有河豚毒素的生物而累积河豚毒素。对此，我们开展了高毒性半褶织纹螺的室内培养实验，以及河豚毒素在不同种类织纹螺体内的累积和排出的模拟实验，并定期采样，通过液相色谱与串联质谱联用技术（LC-MS/MS）对织纹螺体内河豚毒素及其同系物的含量变化情况进行了分析。室内培养实验发现，从连云港赣榆县采集的高毒性半褶织纹螺，在实验初期，体内毒素含量呈下降的趋势，但从 7月上旬开始，毒素含量突然快速上升，与连云港赣榆县野外采集的织纹螺的毒素含量表现出相似的变化趋势。河豚毒素在不同种织纹螺体内的累积和排出的模拟实验发现，通过投喂高毒性的河豚鱼肝脏（毒性为5×103 MU/g），纵肋织纹螺在一段时间内能够快速累积少量的河豚毒素。当停止投喂有毒河豚鱼肝脏后，毒素含量会快速下降。而在曾导致中毒事件的拟半褶织纹螺中，投喂有毒河豚鱼的肝脏后，其体内毒素含量只有缓慢增加。但在投喂无毒的河豚鱼肝脏后，其毒性却出现了快速增加的现象，这与该地区野外样品的毒性变动状况类似。这些发现显示高毒性半褶、拟半褶织纹螺体内的河豚毒素应当不是食物链累积的结果，而可能是由其自身产生。并且，毒素含量的变化具有一定的生物节律，有可能与产卵、繁殖等自然节律相关。 通过对半褶、纵肋和拟半褶织纹螺的研究工作可以认为，产河豚毒素的细菌不是织纹螺体内河豚毒素的主要来源，并且毒素也不是来自其摄食的食物，推测可能主要是由织纹螺自身产生。织纹螺所表现出的河豚毒素含量的季节性变化，极有可能与产卵、繁殖等自然节律相关，这些发现为预防和管理食用织纹螺中毒事件提供了科学依据。但是，本研究并未完全阐明织纹螺体内河豚毒素的来源，对于织纹螺体内河豚毒素的确切来源以及河豚毒素的代谢和转化机制，还有待于更加深入地研究工作。