两种新型白细胞介素-2的热稳定优越性

研制成两种新型白细胞介素-2(新型IL-2),其一为125Ser-IL-2,另一为125Ala-IL-2(即原型IL-2中125位Cys分别被Ser或Ala取代)。两种新型IL-2均已被高度纯化,并已完成了中试,获准临床应用。发现新型IL-2的热稳定优越性。在65℃加热,新型IL-2的稳定性高于原型,失活比原型慢,被保留的活性也较原型多。在0.1％SDS中能使失活的IL-2逐渐恢复一定的活性,也是新型IL-2恢复活性高。同时,比较了其它条件下这三种重组rIL-2的稳定性及SDS对其复性的影响,发现三者之间差异很大。上述结果均证明:125Ala-IL-2的热稳定性最好,125Ser-IL-2次之,原型较新型差。     

两种新型白细胞介素-2的热稳定优越性

研制成两种新型白细胞介素-2(新型Il,-2),其一为125Ser-IL-2,另一为125Ala—Ⅱ2(即原型IL-2中125位cys分别被Ser或Ala取代)。两种新型IL-2均已被高度纯化,并已完成了中试,获准临床应用。发现新型IL-2的热稳定优越性。在65℃加热,新型IL-2的稳定性高于原型,失活比原型慢,被保留的活性也较原型多。在0．1％SDS中能使失活的IL-2逐渐恢复一定的活性,也是新型IL-2恢复活性高。同时,比较了其它条件下这三种重组rIL-2的稳定性及SDS对其复性的影响,发现三者之间差异很大。上述结果均证明：125A1a—IL-2的热稳定性最好,125sER-IL-2次之,原型较新型差。     

白细胞介素和干扰素等免疫活性物质对肿瘤细胞分泌物的拮抗作用

一些免疫活性物,包括基因工程干扰素(rIFN-α1、rIFN-αA rIFN-γ),白细胞介素(rIL-2、IL-3),PHA和ConA等,无论是单独应用还是任选两种联合应用,都具有拮抗小鼠S180肿瘤分泌物的免疫抑制活性的作用。单独应用以rIL-2、IL3的拮抗作用较强:两种物质联合应用的效果均优于单独应用,若免疫活性物质给药先于肿瘤细胞分泌物,则效果更明显。     

大肠杆菌表达的重组白细胞介素-2的初步纯化

本文对大肠杆菌表达产生的重组白细胞介素-2进行了纯化研究。通过比较两种方法制备的rIL-2包含体的纯度,发现用4mol/L脲溶解可溶性细菌蛋白后可使rIL-2包含体纯度达70％;在高浓度变性剂条件下进行凝胶过滤,解决了rIL-2易聚合的问题;结合透析,利用空气氧化形成高活性氧化型rIL-2;经SephadexG-100凝胶过滤,DEAE离子交换等步骤纯化,得到了均一性rIL-2,纯度达98％,比活达4.3×10~6u/mg蛋白,得率为30.8％。     

白细胞介素-6在工程菌分批培养中的高效表达及其纯化

在确定了培养基及pH值的基础上,进一步观察了升温诱导过程中有机酸的产生及其对工程菌E.coli DH5α(pHV-hIL-6)生长和rIL-6表达的影响。当有机酸浓度低于70mmol/L以下时,菌密度达到干重2～3.5g/L之间收菌,rIL-6的表达水平为25％～32％;当有机酸浓度达到70mmol/L以上时,工程菌的生长不受影响,而rIL-6的表达明显受抑制。产生的有机酸以乙酸为主。收集菌体后,经过破菌,分离提纯的包涵体,其rIL-6的纯度可达到70％。用GuHCl缓冲液溶解包涵体,样品稀释后经过Q Sepharose F F柱纯化,可得到纯度达95％以上的rIL-6。采用依赖IL-6的小鼠杂交瘤细胞系7TD1及MTT比色法测定生物活性,rIL-6的比活性为2×10~8U/mg。     

白细胞介素13通过STAT6途径促进瘢痕成纤维细胞株胶原合成

增生性瘢痕和瘢痕疙瘩是一种皮肤纤维化过程,是创伤修复的必然产物。白细胞介素13(IL-13)是与纤维化等多种疾病发生机制密切相关的细胞因子。本文观察重组白细胞介素13(rIL-13)对瘢痕成纤维细胞株(CRL-1762)的胶原合成作用,探讨其作用机制。CRL-1762细胞分为实验组和对照组,实验组加入rIL-13(100μg/L),用细胞计数和HE染色观察细胞增殖和细胞形态;检测细胞培养上清液中羟脯氨酸(Hyp)含量;RT-PCR检测细胞IL-13受体的表达;用Western blot检测STAT6蛋白磷酸化情况。结果显示CRL-1762细胞表达IL-13受体α1;实验组细胞数量显著增加(P0.01),上清液中Hyp含量显著高于对照组(P0.01);IL-13作用CRL-1762细胞2 h后磷酸化STAT6蛋白表达最强,4h后衰减。由此可见,CRL-1762细胞表达IL-13受体α1,rIL-13通过STAT6途径促进CRL-1762细胞胶原合成。     

E.coli HB101(pBV-IL-6)的流加分批培养及其产物纯化

E.coli HB101(pBV-IL-6)工程菌株的表达产物为N端缺失25个氨基酸残基的IL-6衍生物。通过摇瓶试验选定了培养基配方及pH值的控制范围,在5L发酵罐的半连续培养中确定了培养和诱导时间。在此基础上放大到30L发酵罐进行培养,结果表明,菌密度达到5.15g/L(干重),rIL-6衍生物占菌体总蛋白的34.8％。表达产物经过纯化和复性,纯度达95％以上(SDS-PAGE分析),采用依赖IL-6的小鼠杂交瘤细胞株7TD-1和~3H-TdR掺入法测定生物活性,rIL-6衍生物比活性为7.71×10~6U/mg。     

基因工程人白细胞介素－2的生物功能研究

重组白细胞介素－2(rIL－2),能维持T－淋巴细胞及CTLL-2细胞株(IL-2依赖)的增殖达一个月之久,细胞总数增加了近千倍。这种作用能被rJL-2专-的单克隆抗体破坏,显示rIL-2的专一性,rIL-2能增加天然杀伤细胞(NK)的活动达46—96％,但其剂量太高时反而起抑制作用,它能诱异淋巴因子激活的杀伤细胞(Lvmphokine Activated Killier,LAK)的生成,它还能杀伤转移性人肺癌细胞及抗NK的HL－60癌细胞株,其杀伤效率达37.06—54．84％。这说明我们获得的rIL－2在上述生物功能方面与天然lL－2相似。     

rIL-2工程菌诱导阶段细胞再循环培养的研究

为了减少rIL-2工程菌高密度培养时乙酸的积累,在诱导阶段对该工程菌进行细胞再循环培养的研究,比较了细胞再循环补料液、pH、细胞循环培养时间段对工程菌的生长及rIL-2表达的影响。结果表明在菌密度D_(600)为50时,细胞再循环补料液中酵母抽提物与胰蛋白胨浓度为发酵培养基的5倍就能满足rIL-2表达的需求,同时选择诱导后4～6h之间的细胞再循环培养能有效地防止乙酸的过高积累并减少营养物质的损失,有利于rIL-2的表达。根据以上研究结果得到了rIL-2工程菌诱导阶段细胞再循环培养方法,使得在诱导前菌密度D_(600)为50左右时rIL-2的表达水平约为40％。     

表达H5亚型禽流感病毒HA基因和鸡IL-18基因重组禽痘病毒的构建

[目的]获得共表达H5亚型AIV HA基因和鸡IL-18基因的重组禽痘病毒.[方法]将含痘病毒启动子LP2EP2的HA基因和鸡IL-18基因插入到禽痘病毒转移载体pSY681中,获得重组转移载体pSYHA/IL-18.用脂质体将其转染已感染亲本禽痘病毒S-FPV-017株的鸡胚成纤维细胞,使其在鸡胚成纤维细胞内与禽痘病毒基因组发生同源重组,产生表达HA和IL-18的重组禽痘病毒(rFPV-HA-IL-18).在含有X-gal的营养琼脂培养基上进行蓝斑筛选后,对重组禽痘病毒又进行了多次蚀斑克隆.[结果]以重组禽痘病毒DNA为模板,利用HA基因和鸡IL-18基因引物进行PCR,分别扩增出1条约1.7 kb带和1条0.6 kb左右的带.以间接免疫荧光试验、T细胞转化试验和SPF雏鸡免疫接种证实重组禽痘病毒能表达HA和鸡IL-18,并初步证明鸡IL-18增强HA免疫作用.[结论]重组禽痘病毒能表达具有生物学活性的HA和鸡IL-18.     

On the origin of the Hirudinea and the demise of the Oligochaeta

The phylogenetic relationships of the Clitellata were investigated with a data set of published and new complete 18S rRNA gene sequences of 51 species representing 41 families. Sequences were aligned on the basis of a secondary structure model and analysed with maximum parsimony and maximum likelihood. In contrast to the latter method, parsimony did not recover the monophyly of Clitellata. However, a close scrutiny of the data suggested a spurious attraction between some polychaetes and clitellates. As a rule, molecular trees are closely aligned with morphology-based phylogenies. Acanthobdellida and Euhirudinea were reconciled in their traditional Hirudinea clade and were included in the Oligochaeta with the Branchiobdellida via the Lumbriculidae as a possible link between the two assemblages. While the 18S gene yielded a meaningful historical signal for determining relationships within clitellates, the exact position of Hirudinea and Branchiobdellida within oligochaetes remained unresolved. The lack of phylogenetic signal is interpreted as evidence for a rapid radiation of these taxa. The placement of Clitellata within the Polychaeta remained unresolved. The biological reality of polytomies within annelids is suggested and supports the hypothesis of an extremely ancient radiation of polychaetes and emergence of clitellates.     

On the ontogeny of the haptor and the evolution of the Monogenea

Data on the ontogeny of the posterior haptor of monogeneans were obtained from more than 150 publications and summarised. These data were plotted into diagrams showing evolutionary capacity levels based on the theory of a progressive evolution of marginal hooks, anchors and other attachment components of the posterior haptor in the Monogenea (Malmberg, 1986). 5 + 5 unhinged marginal hooks are assumed to be the most primitive monogenean haptoral condition. Thus the diagrams were founded on a 5 + 5 unhinged marginal hook evolutionary capacity level, and the evolutionary capacity levels of anchors and other haptoral attachement components were arranged according to haptoral ontogenetical sequences. In the final plotting diagram data on hosts, type of spermatozoa, oncomiracidial ciliation, sensilla pattern and protonephridial systems were also included. In this way a number of correlations were revealed. Thus, for example, the number of 5 + 5 marginal hooks correlates with the most primitive monogenean type of spermatozoon and with few sensillae, many ciliated cells and a simple protonephridial system in the oncomiracidium. On the basis of the reviewed data it is concluded that the ancient monogeneans with 5 + 5 unhinged marginal hooks were divided into two main lines, one retaining unhinged marginal hooks and the other evolving hinged marginal hooks. Both main lines have recent representatives at different marginal hook evolutionary capacity levels, i.e. monogeneans retaining a haptor with only marginal hooks. For the main line with hinged marginal hooks the name Articulon-choinea n. subclass is proposed. Members with 8 + 8 hinged marginal hooks only are here called Proanchorea n. superord. Monogeneans with unhinged marginal hooks only are here called Ananchorea n. superord. and three new families are erected for its recent members: Anonchohapteridae n. fam., Acolpentronidae n. fam. and Anacanthoridae n. fam. (with 7 + 7, 8 + 8 and 9 + 9 unhinged marginal hooks, respectively). Except for the families of Articulonchoinea (e.g. Acanthocotylidae, Gyrodactylidae, Tetraonchoididae) Bychowsky's (1957) division of the Monogenea into the Oligonchoinea and Polyonchoinea fits the proposed scheme, i.e. monogeneans with unhinged marginal hooks form one old group, the Oligonchoinea, which have 5 + 5 unhinged marginal hooks, and the other group form the Polyonchoinea, which (with the exception of the Hexabothriidae) has a greater number (7 + 7, 8 + 8 or 9 + 9) of unhinged marginal hooks. It is proposed that both these names, Oligonchoinea (sensu mihi) and Polyonchoinea (sensu mihi), will be retained on one side and Articulonchoinea placed on the other side, which reflects the early monogenean evolution. Except for the members of Ananchorea [Polyonchoinea], all members of the Oligonchoinea and Polyonchoinea have anchors, which imply that they are further evolved, i.e. have passed the 5 + 5 marginal hook evolutionary capacity level (Malmberg, 1986). There are two main types of anchors in the Monogenea: haptoral anchors, with anlages appearing in the haptor, and peduncular anchors, with anlages in the peduncle. There are two types of haptoral anchors: peripheral haptoral anchors, ontogenetically the oldest, and central haptoral anchors. Peduncular anchors, in turn, are ontogenetically younger than peripheral haptoral anchors. There may be two pairs of peduncular anchors: medial peduncular anchors, ontogentically the oldest, and lateral peduncular anchors. Only peduncular (not haptoral) anchors have anchor bars. Monogeneans with haptoral anchors are here called Mediohaptanchorea n. superord. and Laterohaptanchorea n. superord. or haptanchoreans. All oligonchoineans and the oldest polyonchoineans are haptanchoreans. Certain members of Calceostomatidae [Polyonchoinea] are the only monogeneans with both (peripheral) haptoral and peduncular anchors (one pair). These monogeneans are here called Mixanchorea n. superord. Polyonchoineans with peduncular anchors and unhinged marginal hooks are here called the Pedunculanchorea n. superord. The most primitive pedunculanchoreans have only one pair of peduncular anchors with an anchor bar, while the most advanced have both medial and lateral peduncular anchors; each pair having an anchor bar. Certain families of the Articulonchoinea, the Anchorea n. superord., also have peduncular anchors (parallel evolution): only one family, the Sundanonchidae n. fam., has both medial and lateral peduncular anchors, each anchor pair with an anchor bar. Evolutionary lines from different monogenean evolutionary capacity levels are discussed and a new system of classification for the Monogenea is proposed.In agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. EditorIn agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. Editor