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1、-分子生物学-第 41 页Parasitology Research Founded as Zeits chrift fr Parasitenkunde Springer-Verlag Berlin Heidelberg 201310.1007/s00436-013-3339-xOriginal PaperConstruction and use of a Trichinella spiralis phage display library to identify the interactions between parasite and host enterocytes旋毛线虫噬菌体展示文库
2、的建立和使用来鉴别寄生虫和宿主的关系Hui Jun Ren1, Ruo Dan Liu1, Zhong Quan Wang1 and Jing Cui1 (1)Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou, 450052, Peoples Republic of China 郑州大学医学院寄生虫室任慧君，刘若丹，王忠全，和崔进 Zhong Quan Wang (Corresponding author)王忠全（通讯作者）Email: Jing Cui (Corresponding aut
3、hor)Email: Received: 15 January 2013Accepted: 1 February 2013Published online: 19 February 2013Abstract 摘要 Although it has been known for many years that Trichinella spiralis 虽然旋毛线虫通过进入小肠的柱状上皮引起感染已经被知道很多年了。但是螺旋虫的被感染的幼虫识别和入侵肠上皮细胞的是通过哪种机制仍然不知道。人们推测寄生虫和宿主间的分子相互作用也许能调节旋毛虫的识别和入侵。initiates infection by pe
4、netrating the columnar epithelium of the small intestine, the mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells (IECs) are unknown. It is speculated that the molecular interactions between the parasite and host enterocytes may mediate the recogniti
5、on and invasion of IECs by T. spiralis. However, no Trichinella proteins that interact with the 然而没有与肠细胞相互作用的毛线虫属蛋白以前被鉴定。这个实验的目的是通过用一个来自旋毛虫肠内的感染的幼虫的信使RNA来构建T7噬菌体展示cDNA文库来鉴别与肠细胞结合的旋毛虫蛋白。接下来五次筛选，测序和生物信息学分析，十种有意义的与常态小鼠肠细胞结合的旋毛虫蛋白被鉴别。enterocytes have been identified previously. The aim of this study was
6、 to identify Trichinella proteins that bind to IECs by screening a T7 phage display cDNA library constructed using messenger RNA from T. spiralis intestinal infective larvae. Following five rounds of biopanning, sequencing, and bioinformatics analysis, ten T. spiralis proteins (Tsp1Tsp10) with signi
7、ficant binding to normal mouse IECs were identified. The results of the protein classification showed that six 这种蛋白质分类的结果展示了旋毛虫蛋白中的六种蛋白（TSP1，钙化运输ATP酶2蛋白；TSP4，卵质酶-1；TSP6，T-复合酶1亚单位；TSP7,糖基水解酶家族47;TSP8，DNAf复制入口MCM3;TSP10，nudix水解酶）通过鉴定的分子功能被解释。proteins (Tsp1, calcium-transporting ATPase 2 protein; Tsp4,
8、 ；ovochymase-1; Tsp6, T-complex protein 1 subunit eta; Tsp7, glycosyl hydrolase family 47; Tsp8, DNA replication licensing factor MCM3; and Tsp10, nudix hydrolase) of these T. spiralis proteins were annotated with putative molecular functions. Out of the six proteins, five have catalytic 这六种酶五中有催化活性
9、，四种有结合活性，一种有运输作用。抗阻止感染的幼虫通过体外的肠细胞的入侵的TSP10的抗体以及有重组噬菌体T7-Tsp10d 被免疫的小鼠证明在成虫中随着旋毛虫肌肉中的幼虫被激活有62.8%的减少。虽然它们的生物功能仍然没有完全知道，但是这些蛋白也许与宿主肠细胞的潜在入侵功能有关。activity, four have binding activity, and one has transporter activity. Anti-Tsp10 antibodies prevented the in vitro partial invasion of IECs by infective larv
10、ae and the mice immunized with the recombinant phage T7-Tsp10 showed a 62.8 % reduction in adult worms following challenge with T. spiralis muscle larvae. Although their biological functions are not yet fully known, these proteins might be related to the larval invasion of host enterocytes. Future e
11、xperiments will be necessary to ascertain whether these proteins play important roles in the recognition 未来的实验是必须的，这些蛋白在识别和入侵宿主肠细胞中是否发挥着重要的作用？这种构建和筛选旋毛虫噬菌体展示文库为寻找与寄生虫和宿主的蛋白的相互作用的识别和入侵有关的候选基因提供了一种新方法。and invasion of host enterocytes. The construction and biopanning of Trichinella phage display librar
12、ies provide a novel approach for searching for candidate genes that are related to invasion and for identifying protein interactions between parasite and host.The nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers JX478226, JX478227, JX4
13、78228, JX478229, JX478230, JX478231, JX478232, JX478233, JX478234, and JX478235.Introduction序言Trichinellosis is a parasitic zoonosis caused by eating raw or undercooked meat contaminated with infective larvae of the nematode genus Trichinella. Human trichinellosis is an emerging/reemerging 旋毛虫病是一种动物
14、源性寄生虫病，是由于食生肉和被线虫属的旋毛虫的幼虫感染的不熟的肉。人类的旋毛虫病是一种新生的或再生的疾病而且已经在世界上的55个国家被报道。通过吃入含有被囊的旋毛幼虫的动物的肉产生感染。disease and has been reported in 55 countries around the world (Cui et al. ; Pozio ; Wang et al. , ). Infection occurs by the consumption of animal meat containing encapsulated Trichinella larvae. Once inges
15、ted, the muscle larvae (ML) are released from their capsules 一旦被摄入，肌肉中的幼虫由于宿主消化酶的作用下在十二指肠中从它们的被囊中释放出来，然后在肠内容物和胆汁中被激活。然后被活化的肠内的被感染的幼虫进入宿主小肠的上皮细胞。不久以后幼虫分化四次（10-28h 感染后，hpi）后成为成虫再进行交配和繁殖，产生下一代的幼虫。in the duodenum as the result of the action of the hosts digestive enzymes and are activated by exposure to
16、 the intestinal contents or bile. Then, the activated intestinal infective larvae penetrate into the epithelial cells of the hosts small intestine. Shortly thereafter, the larvae molt four times (1028 h post infection, hpi) and mature into adults that mate and reproduce, yielding the next generation
17、 of larvae (Kang et al. ). The life cycle of Trichinella spiralis is completed when newborn larvae develop into ML and induce the transformation of muscle cells into nurse cells (Campbell). It is well known that the invasion of the host 当新生的幼虫长成成虫后旋毛虫的一次生活史被完成而且激发肌肉细胞向繁殖细胞转化。已经被证明在旋毛虫感染中通过感染态的幼虫入侵宿主
18、的肠上皮是第一步。先前的研究说明感染的幼虫侵入肠内上皮细胞然后移行至组织上皮，持续的入侵和寄居在新生细胞的细胞质上。intestinal epithelium by infective larvae is the first step inT. spiralis infection. Previous studies have shown that infective larvae invade the intestinal epithelial cells (Dunn and Wright ) and then migrate within the epithelium, continual
19、ly invading and occupying the cytoplasm of new cells (Wright et al. ). To date, the mechanisms by which T. spiralis infective larvae recognize, invade, and migrate within the 迄今为止，旋毛虫感染幼虫的识别，入侵，以及移行到肠上皮的机制和建立它们的多细胞内龛没有被阐明。幼虫不具有口腔附属物或者一个刺突，意味着侵入肠内上皮细胞也许不仅仅只是机械进入的结果但是也许是被表面糖蛋白和口腔分泌物介导的入侵幼虫的一种方式。这些幼虫的蛋
20、白也许与肠内上皮细胞相互作用，也许在入侵肠内上皮细胞的过程中发挥着关键的作用。然而幼虫的蛋白与入侵肠内上皮细胞的关系还没有被鉴定。intestinal epithelium and establish their intramulticellular niche have not been elucidated. The larvae do not possess oral appendages or a spike (Bruce ), implying that the invasion of intestinal epithelial cells (IECs) may not be sim
21、ply a result of mechanical penetration but may be mediated by surface glycoproteins and the oral secretions of the infective larvae (ManWarren et al. ). These larval proteins may interact with IECs and may play a key role during the larval invasion of IECs. However, the larval proteins related to in
22、vasion of IECs have not yet been identified (Nagano et al. ). The use of conventional protein techniques to isolate Trichinella proteins has limited the discovery of new proteins that interact with host cells. Although an epithelial cell model for the in vitro invasion of Trichinella larvae has been
23、 developed (Gagliardo et al. ; ManWarren 用常规的蛋白分离技术区分离旋毛虫蛋白限制了与宿主细胞相互作用的新的蛋白的发现。虽然一种在体外入侵旋毛虫幼虫的肠上皮胞腔模型已经被建立，但是存在与旋毛虫蛋白提取液里的被污染的肠囊肿蛋白使获得纯化的与肠内上皮细胞相互作用的旋毛虫蛋白变得非常困难。另外测定编码与宿主肠细胞相互作用的旋毛虫蛋白的特异基因也是非常困难。为了解决这些限制，噬菌体展示技术被用于研究这些生物分子相互作用中所表现出来的作用。et al. ; Ren et al. ), the presence of contaminating enterocyte
24、 proteins in extracts of Trichinella larval protein makes it extremely difficult to obtain purified Trichinella proteins that interact with IECs. In addition, it is very difficult to determine the specific gene that encodes the Trichinella proteins that interact with host enterocytes. To overcome th
25、ese limitations, phage display technology was used in this study to characterize these biomolecular interactions. Since phage display technique was developed by Smith (), it has been used as a powerful technique for the selection of ligands that bind to any desired target (Paschke ; Xiao et al. ; Gu
26、o et al. ). In the phage display technique, peptides or proteins are expressed on the surface of phages as fusion proteins (Smith ). This allows the selection and amplification of phage clones with specific binding activities. Previous studies using Trichinella phage display libraries自从噬菌体展示技术在1985年
27、被史密斯发现，它已经被用作一种强大的技术用于对必须靶点的配体的选择。运用噬菌体展示技术，肽类和蛋白质作为融合蛋白在噬菌体表面被表达。这就使噬菌体克隆 的选择和放大具有特异的结合活性。先前的研究用旋毛虫噬菌体展示文库主要是关于早期的诊断抗原的研究。然而，就我们现在的技术水平还没有使用噬菌体展示技术去识别旋毛虫蛋白与肠上皮细胞的结合的报道。在这个实验中，我们用一种以噬菌体展示文库为基础的新方法去筛选出与普通小鼠肠内上皮细胞相互作用的未经确认的旋毛虫蛋白。 focused primarily on early diagnostic antigens (Zocevic et al. ). Howeve
28、r, to the best of our knowledge, there has been no report of the use of the phage display technique to identify Trichinella proteins that bind to intestinal epithelial cells. In this study, we used a novel approach based on phage display libraries to screen for unidentified Trichinella proteins that
29、 interact with normal mouse IECs.Materials and methods材料和方法Parasites and experiment animals寄生虫和实验动物 The isolates (ISS534) of T. spiralis used in this study were obtained from a domestic pig in Nanyang City of Henan Province, China. The isolate was maintained by serial passaging in Kunming mice in ou
30、r laboratory. Six-week-old female BALB/c mice were obtained from the 在本实验中被分离出的旋毛虫是从中国河南省南阳市的一家家养猪上获得的。分离出的旋毛虫通过我们实验室的昆明系小鼠身上连续传代。六日龄的雌BALB/c小鼠从河南省实验中心获得（中国，郑州）。Experimental Animal Center of Henan Province (Zhengzhou, China). The mice were maintained under specific pathogen-free conditions with posi
31、tive-pressure filtered air and sterilized food and water. All procedures of animal experiment were approved by the Life Science Ethics Committee of Zhengzhou University (Permission No. SYXK 2007-0009).这些小鼠被饲养在正压的过滤的空气和灭菌的食物和水的无特定病原体的条件下。所有动物实验的操作程序都被郑州大学生命。科学道德委员会批准 ML was recovered from infected mi
32、ce at 42 days post infection by artificial digestion as described previously (Gamble et al. ; Li et al. ; Yang et al. ). After recovery, the larvae were washed three times with normal saline and then orally inoculated into 50 mice, with 5,000 ML per mouse in a volume of 100 l. All the infected mice
33、were euthanized by anesthetic inhalation of isoflurane (Sigma, USA) at 2 hpi. The small intestines were collected, cut along their entire length, and washed in pre-warmed phosphate-buffered saline (PBS) supplemented 肌肉中的幼虫依照以前的表述通过人工消化从被感染后42天的小鼠复苏。复苏后，幼虫用生理盐水洗涤三次，然后通过口服的方式接种50只小鼠，接种量为每100微升体积中含5000
34、个幼虫。所有的小鼠通过2灌注指数的量用异氟醚麻醉剂使其致死。然后收集小肠保留他的整个长度，再用事先加温的添加了抗生素（200 U/ml青霉素和200U/ml链霉素）的磷酸盐缓冲液冲洗。然后，小肠切碎成混合物在300-m的网筛中用PBS在37C培养2h。被释放的感染性幼虫通过一个200-m的网筛过滤和差示沉降30min从肠混合物中被分离。用消过毒的无RNA酶的水洗涤几次后，得到的幼虫在600g下离心10min然后在80 C储存直到使用。with antibiotics (200 U/ml penicillin and 200 g/ml streptomycin). Then, the small
35、 intestines were cut into pieces and incubated in PBS at 37 C for 2 h on a 300-m mesh sieve. The released intestinal infective larvae were separated from intestinal debris by filtration through a 200-m mesh sieve and differential sedimentation for 30 min (Ren et al. ). After several washes in steril
36、e RNase-free water, the larvae were centrifuged at 600g for 10 min and stored at 80 C until use.Intestinal epithelial cells肠上皮细胞In our experiments, normal mouse IECs used were obtained from fetal mouse small intestines and were susceptible to invasion by T. spiralis (Ren et al. ). The IECs were cult
37、ured (5 % CO2, 37 C) in complete DMEM containing 4 mM glutamine, 20 mM Hepes, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 U/ml streptomycin, 0.1 U/ml bovine insulin (Sigma), and 10 % fetal bovine serum (Gibco). The cells在我们的实验中，标准的肠上皮细胞是从胎儿小鼠的小肠获得的，它对旋毛虫的入侵敏感。肠上皮细胞在完全细胞培养基上培养（5 % CO2, 37 C）。包含4mm
38、olL谷氨酰胺，20mmolL羟乙基哌嗪乙硫磺酸，1 mmolL牛胰岛素,和10 %的牛胎儿血清。处理后的肠上皮细胞在中在本实验中的第八步骤被使用。单层细胞通过胰蛋白酶消化作用（在PBS中0.5 %胰蛋白酶，0.54 mmolL EDTA，在 23 C 5 min)被分散were used at passage 8 for the experiment. Cell monolayers were dispersed by trypsinization (0.5 % trypsin0.54 mM EDTA in PBS, at 23 C for 5 min).RNA isolation and
39、construction of a Trichinella phage display library RNA分离和旋毛虫噬菌体展示文库的构造 Total RNA was isolated from the intestinal infective larvae of T. spiralis at 2 hpi using TRIzol reagent according to the manufacturers instructions (Invitrogen). The purified total RNA was quantified with a spectrophotometer (N
40、anodrop) at wavelengths of 230, 260, and 280 nm as described previously (Wang et al.). The integrity of the total RNA was verified by running samples on 1.5 % agarose gels. Then, messenger RNA (mRNA) was purified from the total RNA using an Oligotex mRNA Mini kit (Qiagen). Random-primed first-strand
41、 cDNA was synthesized using 4 g of Trichinella mRNA. After second-strand 肠内具有感染性的旋毛虫幼虫的全部RNA使用厂商构建好的试剂盒以2倍的指数被分离。根据以前的资料全部纯化好的RNA使用分光光度计分别用波长为230,260,280nm的条件下被定量。通过用1.5 %琼脂糖凝胶跑样品全部的RNA完整性被证实。然后，信使RNA用一种Oligotex mRNA Mini kit从total RNA被纯化。随机引物第一链的cDNA用4g旋毛虫mRNA合成。第二条链合成后，cDNA末端用Novagens T7 Select 10
42、-3 Orient Express TM cDNA克隆系统修饰。synthesis, the cDNA ends were modified using Novagens T7 Select 10-3 Orient Express TM cDNA cloning system. Excess linkers and cDNAs shorter than 250 base pairs (bp) were removed by gel filtration. The Trichinella phage display library was created by inserting the mod
43、ified cDNAs into the T7Select 10-3bEcoRI/HindIII vector arms. The recombinant vectors were subsequently packaged with 25 l of T7过量的连接物和短于250个碱基对的cDNAs通过凝胶过滤被移除。旋毛虫噬菌体展示文库通过插入被修饰过的cDNAs到T7Select 10-3bEcoRI/HindIII 载体被创建。这个重组体载体随后用25 l的T7包装提取物被包装然后在大肠杆菌中持续培养。在这个培养过程中融合蛋白被表达然后展示在噬菌体表面。最后，装配好的噬菌体文库用菌斑试验
44、确定效价。然后放大的文库被保存在80 C，10 %的甘油中。Packaging Extracts and propagated in Escherichia coli (strain BLT5403), during which time the fusion proteins were expressed and displayed on the phage surface. Finally, the titer of the packaged phage library was determined using a plaque assay, and the amplified libra
45、ry was stored at 80 C in 10 % glycerol.Biopanning生物淘选The T7 phage display cDNA library from T. spiralis intestinal infective larvae was screened by biopanning against normal mouse IECs. Cells grown to subconfluence (forming a monolayer) in a plastic 96-well ELISA plate were washed once with pre-warm
46、ed culture medium and further incubated for 30 min with culture medium containing 0.1 % bovine serum albumin (BSA). Phages were added to the 96-well culture plate to a final concentration of 23108 plaque-forming unit (pfu)/ml, 这个来自旋毛虫感染性幼虫的cDNA T7噬菌体展示文库在常规小鼠的肠上皮细胞中通过生物淘选被筛选。细胞在96孔的ELISA塑料平板上生长成亚融合状
47、态时（形成一种单层的）用事先预热的培养液洗涤一次然后在含有0.1 %牛血清白蛋白的培养液中培养30 min。噬菌体以23108pfu/ml的终浓度被加入到96孔的ELISA塑料平板上。然后这个平板在37 C条件下被培养2 h。这种细胞含有0.05 % Tween-20的PBS洗涤五次，然后细胞边界噬菌体在室温下用含有4molL的200 l PBST 20 min被洗脱。and the plate was incubated for 2 h at 37 C. The cells were washed five times with PBS containing 0.05 % Tween-20
48、(PBST), and then the cell surface-bound phages were eluted for 20 min at room temperature by adding 200 l of PBST containing 4 M urea. The obtained phages were amplified overnight at 37 C in 50 ml of early log-phase E. coli BLT5403 cells. Following amplification, the lysed culture was centrifuged at 8,000g for 10 min, and the supernatant was used for the next round of biopanning. A total of five rounds of biopanning were performed. The 获得的噬菌体在37 C条件下的用50 ml的大肠杆菌BLT5403细胞的早期对数期隔夜进行扩增。扩增后溶解的培养物以每10 min，8,000g的转速被离心，然后上清液被用于下一轮的筛选。五轮筛选完成。每轮筛选使用相同数量的噬菌体（2107 个噬菌体）。经过五轮的筛选后，最后洗脱的噬菌体被弃掉。然后DNA