【作者】 杜欣军；

【导师】 王金星；

【作者基本信息】 山东大学 ， 动物学， 2007， 博士

【摘要】 中国明对虾主要分布于我国黄渤海和朝鲜西部沿海，是我国最具经济价值和营养价值的海产品之一。大量捕捞和各种病原体(细菌、病毒等)的感染，严重威胁着这一物种的生存。自上世纪七十年代以来在我国沿海一带，开展了大量的中国明对虾养殖生产，使得养殖产量大大超过海捕量。但是自从1993年大规模爆发对虾流行病(对虾白斑综合症)以来，中国明对虾的养殖遭到重创，产量一直没有恢复。这一方面与白斑综合症病毒的高度感染性和致死性有关，另一方面也与中国明对虾自身免疫力有关。因此，对于中国明对虾免疫系统的研究显得尤为重要。在理论上可以加深我们对于无脊椎动物先天免疫的认识，丰富先天免疫的理论；在实际应用中，可以为中国明对虾的疾病防治和提高抗病力提供新思路和新方法。本研究通过淘选噬菌体展示文库、同源克隆和抑制性消减杂交的方法从中国明对虾中分别克隆得到了围食膜蛋白基因、脂多糖-β-1，3-葡聚糖结合蛋白(LGBP)和三种抗菌肽(crustin)基因，并对这些基因的性质做了进一步研究。这些研究使我们对中国明对虾免疫的模式识别和效应分子有了进一步的认识。一、中国明对虾围食膜蛋白基因克隆和性质研究我们用弧菌和金黄色葡萄球菌刺激中国明对虾以后，提取其血细胞的总RNA。再提取mRNA后用其构建了T7噬菌体展示文库。选择了多种配体来进行文库淘选，在用大肠杆菌经过3轮淘选以后获得了一段围食膜蛋白的基因。在这一片段的基础上设计基因特异性引物利用RACE和SMART cDNA技术获得了基因的全长。中国明对虾围食膜蛋白基因(Fc-PTPH)全长为1028bp，开放阅读框为822bp，共编码274个氨基酸。基因的5’端含有17bp的非编码区，3’端含有209bp的非编码区。这一基因编码的蛋白为30.6kDa，其理论等电点为4.92。蛋白前20个氨基酸为信号肽序列，其含有4个围食膜蛋白A样结构域(peritrophin A-like domains)，经过SMART预测，其中后三个为几丁质结合结构域(chitin-binding domains，CBDs)。通过Northern blot和RT-PCR研究发现，Fc-PTPH组成性高表达于卵巢中，在肝胰腺中没有表达，而在其它组织(血细胞、心、胃、鳃、肠、精巢)中细菌刺激才能诱导这一基因的表达。我们还用Northern blot方法研究了Fc-PTPH在肠和卵巢中细菌刺激后不同时间的表达变化。结果显示，在肠中刺激16小时后能够检测到基因的表达，在24小时时表达量达到最高。而在卵巢中，在刺激后不同时间Fc-PTPH的表达没有变化，一直维持着高表达模式。原位杂交显示这一基因的mRNA存在于颗粒血细胞和卵母细胞的细胞质中。设计了成熟肽表达引物，经过PCR扩增后连入pET-30a(+)载体，转化大肠杆菌BL21(DE3)进行原核表达。由于表达产物形成包涵体，所以只有经过变性复性后，才能利用His-Bind亲和层析柱进行纯化。利用纯化的蛋白作为抗原刺激家兔获得了多克隆抗血清，免疫双扩散显示抗体滴度可以达到8倍稀释。Western blot检测也能获得清晰、特异的条带。利用重组的围食膜蛋白进行几丁质结合实验，BSA为对照。结果显示重组的围食膜蛋白具有很强的几丁质结合能力，而BSA完全不能结合几丁质。我们还选择了大肠杆菌和金黄色葡萄球菌验证围食膜蛋白结合细菌的能力，结果显示围食膜蛋白能够结合革兰氏阴性菌，而对革兰氏阳性菌则没有结合能力。二、中国明对虾脂多糖-β-1，3-葡聚糖(LGBP)基因克隆和性质研究根据相近物种的同一类基因设计了两对兼并引物，巢式PCR后获得了中国明对虾LGBP基因(Fc-LGBP)的中间片段，再根据这一片段设计引物从血细胞SMART cDNA中克隆得到了基因全长。基因全长为1253bp，包含1101bp的开放阅读框。在基因的5’端含有7bp的非编码区，3’端含有129bp的非编码区。这一基因编码的蛋白质由366个氨基酸构成，其中前17个氨基酸为信号肽序列。预测成熟肽分子量为39.8kDa，等电点为4.43。通过软件预测，中国明对虾LGBP含有两个N糖基化位点和一个细胞粘连位点(Arg-Gly-Asp，RGD)。从蛋白94位氨基酸至312位氨基酸是一个糖基水解酶结构域。选取正常及细菌刺激的血细胞、心、肝胰腺、胃、鳃、肠的总RNA进行Northern blot分析，结果显示Fc-LGBP仅表达于血细胞中，细菌刺激24小时后基因表达量下降。这一结果在RT-PCR中得到了进一步验证，但是由于RT-PCR更灵敏一些，还可以在肝胰腺和鳃中检测到基因的表达，但是其表达很弱。我们选取了多种组织来研究Fc-LGBP基因表达的组织定位，原位杂交显示Fc-LGBP基因的mRNA只存在于颗粒细胞的细胞质中。根据基因序列设计成熟肽表达引物，将基因片段克隆到载体pET-30a(+)中，转化其感受态细胞进行原核表达。重组表达的蛋白也形成包涵体，变性、复性以后用His-Bind亲和柱进行纯化。利用重组表达并纯化的LGBP作为抗原免疫家兔，9周后获得多克隆抗血清。在获得了LGBP多克隆抗体以后，我们研究了LGBP蛋白在不同组织中的分布。首先利用免疫组化的方法研究了LGBP的组织定位，一抗反应后，再用FITC标记的羊抗兔IgG检测，在荧光显微镜下可以观察到LGBP蛋白定位于绝大多数血细胞的细胞膜上，形成颗粒状的荧光信号。利用多克隆抗体，我们还研究了LGBP蛋白在对虾各组织中的表达模式。将对虾各组织(血浆、血细胞、心、肝胰腺、鳃)蛋白进行SDS-PAGE并转膜后，用LGBP多克隆抗体进行标记，再用辣根过氧化物酶标记的羊抗兔IgG进行二抗标记，显色后发现，天然的LGBP蛋白主要存在于血细胞中，在肝胰腺也有较弱的表达，天然蛋白的分子量约为40kDa。在其它组织中则没有检测到蛋白的存在。最后，我们选取了9种微生物进行了结合实验，包括：2种革兰氏阴性菌(大肠杆菌，Escherichia coli；肺炎克氏杆菌，Klebsiella peneumoniae)，5种革兰氏阳性菌(金球菌，Staphylococcus aureus；苏云金芽孢杆菌，Bacillus thuringiensis；藤黄微球菌，Micrococcus luteus；蜡状芽孢杆菌，Bacillus cereus；巨大芽孢杆菌，Bacillus megaterium)和一种酵母(毕赤酵母，Pichia pastoris)。结果显示Fc-LGBP对革兰氏阴性菌(K．peneumoniae)的结合能力最强。对革兰氏阴性菌(E．coli)、革兰氏阳性菌(M．luteus，B．megaterium)和酵母(P．pastoris)的结合能力稍弱。而对于其它的革兰氏阳性菌(S．aureus，B．thuringiensis or B．cereus)则没有结合能力。三、中国明对虾三种甲壳肽(crustin)类抗菌肽的基因克隆和性质研究我们在中国明对虾血细胞抑制性消减杂交(suppression subtractive hybridization，SSH)文库测序的EST中发现了一段序列与crustin基因相似，根据这段序列设计特异性前引物从血细胞SMART-cDNA中获得了基因的3’端，又设计特异性后引物，获得了基因5’端的部分序列，将此基因命名为Fc-crustinⅠ。在扩增Fc-crustinⅠ的5’端时，获得的片段与已经获得的Fc-crustinⅠ的部分序列除引物外不能完全重叠，便命名为Fc-crustinⅡ；根据这一基因片段设计基因特异性引物，获得了Fc-crustinⅡ基因的完整序列。在Fc-crustinⅠ3’端克隆时，在卵巢组织中获得的片段与预期大小不一致，将其克隆测序后是一条具有完整的3’端的EST，BLAST结果显示其与crustin具有较高的同源性，便命名为Fc-crustinⅢ；根据Fc-crustinⅢ的这一序列设计引物获得了这一基因的全序列。已经获得的Fc-crustinⅠ部分基因序列含有完整的乳清酸性蛋白(whey acidic protein，WAP)结构域，位于基因的3’端由49个氨基酸构成。Fc-crustinⅡ基因全长473bp，包含一个390bp的开放阅读框，编码130个氨基酸。在基因的5’端有4bp的非编码序列，3’端的非编码序列为79bp。通过ExPASy网站在线预测，这一基因编码的蛋白分子量为14.0kDa，其理论等电点为8.60。蛋白前17个氨基酸为信号肽序列，蛋白C端83-124位氨基酸形成WAP结构域。Fc-crustinⅢ基因全长为574bp，其开放阅读框为462bp，共编码154个氨基酸。基因5’端和3’端的非编码区分别为38bp和74bp。通过软件预测Fc-crustinⅢ基因编码的蛋白分子量为17.79kDa，其理论等电点为4.64。前20个氨基酸形成信号肽，62-127位氨基酸构成WAP结构域。选取正常及细菌刺激24小时中国明对虾血细胞、心、肝胰腺、胃、鳃、肠、卵巢和精巢组织，利用RT-PCR方法分析三种crustin基因的表达模式。结果显示Fc-crustinⅠ基因广泛表达于各种组织，在血细胞中表达量最高，精巢中表达量最低。在多数组织中细菌刺激前后Fc-crustinⅠ表达变化不大，但是在胃中刺激后表达上调比较明显。Fc-crustinⅡ也广泛表达于各种组织中，在各组织中的表达差异与Fc-crustinⅠ相似，在血细胞中的表达量最高。细菌刺激后在心、鳃、卵巢组织中表达上调比较明显，而在其它组织中，刺激前后表达变化不大。Fc-crustinⅢ的表达模式同Ⅰ和Ⅱ差别比较大，Fc-crustinⅢ只表达于少数几种组织中，包括正常心、刺激胃、正常卵巢和刺激卵巢。另外，在刺激的心和肠中有少量表达。在其余组织中则没有检测到Fc-crustinⅢ基因的表达。设计成熟肽表达引物，将Fc-crustinⅡ和Fc-crustinⅢ分别克隆入pET-30(+)，然后转入大肠杆菌进行原核表达。利用His-Bind亲和柱纯化以后检测其抑菌活性，结果表明原核重组表达的Fc-crustinⅡ和Fc-crustinⅢ的成熟肽对革兰氏阳性菌和革兰氏阴性菌都没有抑制活性。又重新设计表达前引物，将信号肽一起进行原核重组表达，用管碟法检测后，Fc-crustinⅢ重组表达蛋白的抑菌活性没有检测到，而Fc-crustinⅡ对藤黄微球菌和金黄色葡萄球菌两种革兰氏阳性菌具有抑制活性。总之，本研究成功从中国明对虾中克隆到围食膜蛋白基因、LGBP基因和crustin抗菌肽基因，并研究了三类基因的性质，这些研究使我们对中国明对虾免疫的模式识别和效应分子有了进一步的认识。更多还原

【Abstract】 Chinese shrimp (Fenneropenaeus chinensis) was mainly distributed in Yellow and Bo sea of China and western coast of Korean Peninsula as an economically and nutritionally important shrimp. Excessive capture and infection by many pathogens such as bacteria and virus seriously embarrassed survival of the animal. Recent years, marine aquaculture of F. chinensis was carried out widely around the coast of China and so the production of cultured animals were far more than the captured ones. However shrimp cultivation has been suffering serious problems due to the outbreak of the white spot syndrome since 1993. The high mortality of F. chinensis in cultivation was either due to the serious infectious activity of the the white spot syndrome virus or to the low immune activity of the animal. So, it is necessary to study the immune system of F. chinensis. This investigation could make us know more about the immune defence mechanisms of invertebrate. On the other hand, knowledge on immune system of shrimp could be helpful in F. chinensis cultivation through improving its immune activity and consequently reduce economical loss.The panning of T7 phage display library, homology cloning and suppression subtractive hybridization (SSH) were used to obtain immune related genes in the studies. A peritrophin, a lipopolysaccharide andβ-1,3-glucan binding protein (LGBP) and three crustin genes were cloned from F. chinensis, and characterized.1. Identification and molecular characterization of a peritrophin-like proteinfrom fleshy prawn (F. chinensis)T7 phage display library was constructed with mRNA from S. aureus and Vibrio anguillarum challenged shrimp. After three rounds of panning a fragment of peritrophin gene was obtained from the library. Modified SMART (Switching Mechanism At 5’ end of RNA Template) cDNA and the long distance PCR (LD-PCR) procedures were used to complete the 5’ and 3’ ends of the gene with gene-specific primers and adaptor primers. The full length of the cDNA of peritrophin of F. chinensis (Fc-PTPH) was 1048 bp including an 822 bp open reading frame encoding a 274-residue protein. There was a 17 bp untranslated region in the 5’ end and a 209 bp untranslated region in the 3’ end. The molecular weight of the deduced protein was 30.6 kDa and the theoretical pI was 4.92. The deduced protein, with a signal peptide (1-20 residues), contained four peritrophin A-like domains and the latter three domains was chitin-binding domains (CBDs) (residues 83-145, 146-199 and 201-258) by SMART analysis.The results of Northern blot and RT-PCR demonstrated that Fc-PTPH constitutively expressed in ovaries but did not express in hepatopancreas at all. Otherwise, in other tissues including haemocytes, heart, stomach, gills, intestine and spermary the gene was induced expression. The time course expression patterns of Fc-PTPH after bacteria-challenge were examined in intestine and ovary with Northern blot. The results showed that Fc-PTPH is up-regulated in intestines after bacterial challenge: expression of the gene was detected by 16 h after infection, and at 24 h post-infection the transcription of the gene reached the highest level. A very strong signal was detected in ovaries and the signal intensity did not change between the 4-24 h infection periods. The mRNA of Fc-PTPH was clearly located in the cytoplasm of oocytes of ovaries and a fraction of the hemocytes in challenged shrimp.Basing on full sequence a pair of primers were synthesized to amplify the mature Fc-PTPH cDNA fragment. Then the fragment was subcloned into pET-30(+) vector and the recombinant plasmid was transformed into competent cells of E.coli BL21(DE3) for recombinant expression. Because inclusion bodies were formed denaturation and refolding were introduced before purification with His Bind resin chromatography. Polyclonal antiserum was obtained by injection purified recombinant expressed FC-PTPH (rFC-PTPH) into rabbit and the titer and specificity was examined with double immunodiffusion and Western blot.Binding activities of rFC-PTPH was analyzed and the results indicated that rFC-PTPH could tightly bind to chitin and E.coli but could not bind to 5. aureus at all.2. Molecular cloning and characterization of a lipopolysaccharide andβ-1, 3-glucan binding protein from fleshy prawn (F. chinensis)Two pairs of degenerate primers were designed to obtain a fragment of the LGBP gene from haemocytes of F. chinensis (Fc-LGBP) utilizing nested PCR and then the full length cDNA was obtained with modified SMART cDNA and the LD-PCR procedures. The full-length LGBP cDNA fragment (1253 bp) of F. chinensis was obtained by overlapping three cDNA fragments and included a 7 bp 5’ untranslated region (UTR), a 1101 bp open reading frame and a 129 bp untranslated region in the 3’ UTR with a 16 bp polyA tail. The ORF encoded a 366 amino acid protein with a 17 amino acid signal peptide. The mature Fc-LGBP, therefore, consisted of 349 amino acid residues with a calculated molecular mass of 39.8 kDa and a predicted isoelectric point of 4.43. The mature protein included two putative glycosylation sites(Asn-Xaa-Ser) for N-linked carbohydrate chains and a putative cell adhesion site (integrin binding site), Arg-Gly-Asp (RGD). Additionally, a glycosyl hydrolase domain was identified (Ser 94 to Ala 312).The expression profiles of bacteria-challenged haemocytes, heart, hepatopancreas, stomach, gills and intestine were analyzed by Northern hybridization. The result demonstrated that Fc-LGBP only expressed in haemocytes and was down-regulated by bacteria-challenge post 24 h injection. This result was confirmed by RT-PCR. Because of more sensitivity of RT-PCR gene expression could also be detected in hepatopancreas and gills although the signals were weak. Utilizing in situ hybridization, we investigated a variety of tissues types to localize Fc-LGBP mRNA in challenged fleshy prawn. Our data suggested that hybridized signal was only detected in haemocytes, with strong signals noted in the cytoplasm of granular haemocytes.The sequence coding for mature protein was amplified and ligated into expression vector pET30a(+) . The recombinant vector was transformed into competent E. coli BL21(DE3) host cells. After denature and refolding the recombinant FC-LGBP (rFC-LGBP) was purified with His Bind resin chromatography. Polyclonal antiserum was obtained from rabbit after continuous injection of rFC-LGBP.With polyclonal antibodies we located FC-LGBP in haemocytes. Goat anti-rabbit IgG conjugated with FITC was used as second antibody. The result confirmed FC-LGBP membrane localization in most haemocytes, with granule-like signals noted in some areas of haemocyte membrane. With polyclonal antibodies we also studied the nature protein in different tissues including plasm, haemocytes, heart hepatopancreas, gills. After incubation with second antibody (HRP-conjugated goat anti-rabbit IgG) and visualization the natural FC-LGBP was characterized as a 40kDa protein in haemocytes and hepatopancreas. But in other tissues no signal could be detected at all.Gram-negative bacteria (E. coli; Klebsiella peneumoniae), Gram-positive bacteria (S. aureus; Bacillus thuringiensis; Micrococcus luteus; Bacillus cereus; Bacillus megaterium) and yeast (Pichia pastoris) were utilized to analyze FC-LGBP binding activity. Our data suggested that FC-LGBP proteinwas able to strongly bind to Gram-negative bacteria (K. peneumoniae) and relatively less to E. coli, Gram-positive bacteria (M. luteus, B. megaterium) and yeast (P. pastoris).We noted no binding activity to S. aureus, B. thuringiensis or B. cereus.3. Molecular cloning and characterization of three crustin-like proteins from fleshy prawn (F. chinensis)In the haemocyte SSH library of Chinese shrimp a fragment of a crustin-like gene was obtained. With gene specific primers, the 3’ end of the crustin cDNA was obstained from SMART-cDNA and the gene was named Fc-crustin I. During cloning the 5’ end of Fc-crustin I a crustin-like fragment that did not overlap with Fc-crustin I very well was obstained and named Fc-crustin II. A gene specific primer was synthesized to get the full cDNA of Fc-crustin II gene. In the result of 3’ end cloning of Fc-crustin I a band other than the expected one was detected. The fragment exhibited high similarity with crustin-like protein and then named Fc-crustin III. The full cDNA was also got from SMART-cDNA with a gene specific primer.There was a complete WAP (whey acidic protein) domain in the obtained fragment of Fc-crustin I. The domain was near the C terminal of the protein and composed of 49 amino acid residues.The full length of the cDNA of Fc-crustin II of F. chinensis was 473 bp including a 390 bp open reading frame that encoding 130 amino acids. There was a 4 bp untranslated region in the 5’ end and a 79 bp untranslated region in the 3’ end. The molecular weight of the deduced protein was 14.0 kDa and the theoretical isoelectric point was 8.60. The former 17 amino acids formed a signal peptide. The deduced protein also contained a WAP domain (residues 83-124).The full-length Fc-crusin III cDNA of F. chinensis was obtained by overlapping two cDNA fragments and composed of 574 bp including a 38 bp 5’ untranslated region (UTR), a 462 bp open reading frame and a 74 bp untranslated region in the 3’ UTR1. The ORF encoded a 154 amino acid protein with a 20 amino acid signal peptide. The deduced WAP domain was compsed of residues from 62 to 127. The molecular weight of the deduced protein was 17.79 kDa and the pI was 4.64.Haemocytes, heart, hepatopancreas, stomach, gills, intestine, ovary and spermary of normal and bacteria challenged F. chinensis were selected to study the expression patterns of three crustin-like genes with RT-PCR method. The data demonstrated that Fc-crustin I expressed in all the selected tissues at different levels. There was a strongest signal in haemocytes and weakest signal in spermary. Bacteria-challenge could not change expression levels of the gene evidently in most tissues. But the expression of Fc-crustin I was up-regulated obviously in stomach. Expression pattern of Fc-crustin II was similar to that of Fc-crustin I. The gene also expressed in all the tissues and the highest expression level was also detected in haemocytes. After bacteria-challenge expression of Fc-crustin II was up-regulated obviously in heart, gills and ovary, but there were no obviously changes in other tissues. The exprssion pattern of Fc-crustin III differ distinctly from the former two genes. The signals could only be detected in some tissues including normal heart, ovary and challenged stomach, ovary. Otherwise there were weak signals could be detected in challenged heart and intestine.The cDNA fragments encoding for mature proteins of Fc-crustin II and Fc-crustin III were subcloned into pET30a(+) and expressed in E. coli. The mature proteins purified with His Bind resin chromatography were used to analyze the antimicrobial activities against Gram-positive and Gram-negative bacteria. But no activities could be detected. And then the fragments coding for all the amino acids including signal peptide were amplified with new primers and expressed in E. coli. The antimicrobial activities of recombinant expressed FC-crustin III could not be detected. But the antimicrobial activities of recombinant expressed FC-crutin II against two Gram-pssitive bacteria including Micrococcus luteus and Staphylococcus aureus could be detected with cylinder-plate method.In conclusion, Peritrophin, LGBP and crustin genes had been successfully cloned from Chinese shrimp and their functions and features were characterized. These works accumulated our knowledge on immune system of shrimp.更多还原