栉孔扇贝（Chlamys farreri）组织凝集活性分析及一种C-型凝集素单克隆抗体的研制和特性研究
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摘要
凝集素是贝类生物体液免疫的主要执行者之一，是抵御外来病原体侵袭和环境刺激的重要“屏障”，在病原识别以及止血、凝固、物质运输和创伤修复等生理过程中都具有重要作用。C-型凝集素（C-type lectin）作为宿主防御系统中的一种模式识别蛋白（Pattern recognition protein，PRP），在病原体识别和细胞间的相互作用上具有重要作用。本论文比较了栉孔扇贝血淋巴以及其它不同组织粗提液对高等动物血细胞和多种菌体的凝集活性；并从栉孔扇贝血淋巴中分离纯化了甘露聚糖结合凝集素（Mannan-binding lectin，MBL）；制备了C-型凝集素CfLec290的单克隆抗体，并结合RT-PCR技术、原位杂交技术对其在栉孔扇贝（Chlamysfarreri）血细胞以及其它不同组织中的分布特性进行分析，为研究栉孔扇贝凝集素在免疫防御机制中作用积累了数据。本论文主要包括以下4个部分：
（1）栉孔扇贝血淋巴以及其它不同组织粗提液的凝集活性分析。本论文通过超声波破碎或组织匀浆的方式分别制备了栉孔扇贝血细胞以及其它组织，包括鳃、外套膜、肝胰腺、性腺、肾脏以及肌肉组织的蛋白粗提液，并将其与血淋巴上清的蛋白浓度都调整为1mg/mL，然后分别测定这8组样品对2种动物红细胞血液和9种菌悬液的凝集活性，并测定EDTA、多种糖溶液对其凝集活性的影响。结果发现：血淋巴上清不仅可以凝集小鼠红细胞，还可以凝集鳗弧菌、金黄色葡萄球菌、枯草芽孢杆菌以及毕赤酵母，其凝集活性可以被EDTA、葡聚糖、甘露糖-6-磷酸、N-乙酰葡萄糖胺，甘露聚糖、酵母聚糖、硫酸软骨素、肽聚糖和脂多糖不同程度的抑制；鳃组织粗提液可以凝集金黄色葡萄球菌、枯草芽孢杆菌和毕赤酵母，且凝集活性可以被EDTA、N-乙酰半乳糖胺、甘露聚糖、酵母聚糖、硫酸软骨素、肽聚糖和脂多糖不同程度的抑制；外套膜、肝胰腺、性腺和肾脏提取液均只能凝集枯草芽孢杆菌，且该凝集反应主要受到甘露聚糖、酵母聚糖以及肽聚糖的抑制；此外，栉孔扇贝血细胞及肌肉组织粗提液并未发现凝集活性。这些结果表明，在栉孔扇贝体内有多种类型凝集素的存在，且在血淋巴上清中的类型和数量最为丰富，而这正与扇贝强大的体液免疫系统息息相关。
（2）栉孔扇贝血淋巴甘露聚糖结合凝集素的分离纯化。本论文依次通过硫酸铵盐析和甘露聚糖-亲和层析柱亲和层析，从栉孔扇贝血淋巴上清中分离纯化出甘露聚糖结合凝集素，通过对其凝集活性的检测以及蛋白分子量的分析发现：提纯的MBL可以凝集鳗弧菌和毕赤酵母，对高等动物血细胞以及其它检测菌体并无凝集活性；MBL在Native-PAGE中的分子量为645kDa，在SDS-PAGE中的分子量为73kDa，表明该凝集素是由一种亚基组成的多聚体。
（3）抗栉孔扇贝C-型凝集素Cflec290单克隆抗体的制备和筛选。本论文首先通过体外重组表达获得重组蛋白rCfLec290，免疫小鼠后，经细胞融合技术制备了抗rCfLec290单克隆抗体，并分别通过ELISA技术和Western blotting技术对抗体进行筛选，最后筛选出3株可稳定表达单抗的细胞株1A2、1A7和2B6。
（4）C-型凝集素Cflec290在栉孔扇贝血细胞及其它不同组织中的分布。首先通过半定量RT-PCR技术检测Cflec290mRNA在血细胞以及其它不同组织中的表达含量，再通过制备地高辛标记的Cflec290的cRNA核酸探针，检测血细胞滴片以及其它不同组织切片中mRNA表达的位置，最后通过抗重组蛋白rCfLec290单克隆抗体检测该凝集素在血细胞滴片及不同组织切片中的分布情况。结果发现：通过半定量RT-PCR，Cflec290在血细胞以及其它各组织中均有表达，但在外套膜、肾脏以及性腺中的转录水平最高，血细胞、鳃和肝胰腺次之，在肌肉中的转录水平较弱；原位杂交显示，阳性信号主要发现在栉孔扇贝的外套膜表皮细胞以及肝胰腺管壁细胞中，在其它组织中并未发现；通过利用抗重组蛋白rCfLec290的单克隆抗体的间接免疫荧光检测发现，CfLec290蛋白主要分布在扇贝血细胞膜，以及鳃、外套膜、肝胰腺等组织表皮和腔体表面。结果表明，Cflec290可在栉孔扇贝血细胞、外套膜表皮细胞和肝胰腺管壁细胞中合成与表达，功能蛋白则广泛分布于扇贝血细胞及各组织器官中，在血细胞膜，鳃、外套膜、肝胰腺表皮，腔体表面尤为丰富。依此推测，血细胞、外套膜和肝胰腺在Cflec290合成和表达过程中发挥着重要作用，该凝集素在血细胞、外套膜或肝胰腺细胞内合成后，被分泌到细胞外，伴随着体液流动，被运输到全身多组织器官中行使功能。
Lectins contribute a lot to the scallop humoral immune system for avoidingthe pathogen invasion and environmental stimulus. They play an important role in thepathogen recognition, as well as the physiological process of hemostasis, coagulation,material transport and wound healing in scallops. In this dissertation, the agglutinationactivities of the cell-free haemolymph (CFH) and haemocyte, gills, mantle,hepatopancreas, gonad, kidney, adductor muscle of C. farreri were tested separatelyagainst the rabbit blood cells (RBC), mouse blood cells (MBC) and the fluoresceinisothiocyanate-labelled microbes, and a mannan-binding lectin (MBL) was purifiedfrom CFH by affinity chromatography.; and then, monoclonal antibodies wereproduced, and they were used to analyzed the CfLec290distribution characteristics ofC. farreri haemocytes as well as other organizations combined with RT-PCR and insitu hybridization.
(1) Agglutination activities of haemolymph and tissue extracts in scallop C.farreri were tested. In this dissertation, cell-free haemolymph (CFH) and crudeextracts from the haemocytes, gills, mantle, hepatopancreas, gonad, kidney andadductor muscle of C. farreri were obtained, and their agglutination activities weretested separately against RBC, MBC and fluorescein isothiocyanate-labeled microbes.Agglutination inhibition were carried out with EDTA and saccharides. The resultsshowed the CFH could aggregate Vibrio anguillarum, Staphylococcus aureus,Bacillus subtilis, Pichia pastoris and MBC, and the agglutination could be inhibitedby DEAE dextran, EDTA, D-mannose-6-phosphate (M6P), N-acetyl glucosamine(GlcNAc), mannan, zymosan A, chondroitin sulphate, peptidoglycan (PGN) andlipopolysaccharides (LPS); Gill extracts aggregated S. aureus, B. subtilis and P.pastoris, and the agglutination activity could be inhibited by EDTA, N-acetyl galactosamine (GalNAc), mannan, zymosan A, chondroitin sulphate, PGN and LPS;Mantle, hepatopancreas, gonad and kidney extracts could only aggregate B. subtilis,and this could be inhibited by mannan, zymosan A and PGN; however, noagglutination activity was detected using haemocyte or adductor muscle extracts. Theresults indicated that multiple types of lectins exist in C. farreri, and CFH has thehighest agglutination activity.
(2) Mannan-binding lectin (MBL) was purified from haemolymph of C.farreri.MBL was purified from CFH by affinity chromatography using a mannan-sepharosecolumn and analyzed using native-and denaturing-PAGE. The results showed that thepurified MBL could aggregate Vibrio anguillarum, and exhibited a molecular mass of645kDa using native-PAGE and73kDa using SDS-PAGE, suggesting that C. farreriMBL is an polymer composed of one subunit.
(3) Production of the monoclonal antibodies against rCfLec290. Therecombinant protein rCfLec290were produced and purified in vitro. And then thepurified rCfLec290was used to immunize the mouse, and after the cell fusion, ELISAand western blotting were employed to screen the fusion cells, Finally,3hybridomaswere achieved, MAb1A2,1A7and2B6.
(4) Distribution of C-type lectin Cflec290in C. farreri were detected. First,Semi-quantitative RT-PCR and in situ hybridization was employed to detect theCflec290mRNA distribution, and the monoclonal antibodies against rCfLec290wereproduced to detect the distribution of the protein CfLec290in different tissue by IIFAand western blotting. The results showed that: In semi-quantitative RT-PCR, Cflec290mRNA expressed at the highest level in mantle, kidneys and gonads, moderate in gillsand adductor muscles, least in hepatopancreas; In ISH, positive signals were mainlyobserved in the epithelium of mantle and hepatopancreas cells; In IIFA, the positivefluorescent signals were mainly distributed in the membrane of haemocytes, and theedge of gills, mantle, kidney and glands.
（1）栉孔扇贝血淋巴以及其它不同组织粗提液的凝集活性分析。本论文通过超声波破碎或组织匀浆的方式分别制备了栉孔扇贝血细胞以及其它组织，包括鳃、外套膜、肝胰腺、性腺、肾脏以及肌肉组织的蛋白粗提液，并将其与血淋巴上清的蛋白浓度都调整为1mg/mL，然后分别测定这8组样品对2种动物红细胞血液和9种菌悬液的凝集活性，并测定EDTA、多种糖溶液对其凝集活性的影响。结果发现：血淋巴上清不仅可以凝集小鼠红细胞，还可以凝集鳗弧菌、金黄色葡萄球菌、枯草芽孢杆菌以及毕赤酵母，其凝集活性可以被EDTA、葡聚糖、甘露糖-6-磷酸、N-乙酰葡萄糖胺，甘露聚糖、酵母聚糖、硫酸软骨素、肽聚糖和脂多糖不同程度的抑制；鳃组织粗提液可以凝集金黄色葡萄球菌、枯草芽孢杆菌和毕赤酵母，且凝集活性可以被EDTA、N-乙酰半乳糖胺、甘露聚糖、酵母聚糖、硫酸软骨素、肽聚糖和脂多糖不同程度的抑制；外套膜、肝胰腺、性腺和肾脏提取液均只能凝集枯草芽孢杆菌，且该凝集反应主要受到甘露聚糖、酵母聚糖以及肽聚糖的抑制；此外，栉孔扇贝血细胞及肌肉组织粗提液并未发现凝集活性。这些结果表明，在栉孔扇贝体内有多种类型凝集素的存在，且在血淋巴上清中的类型和数量最为丰富，而这正与扇贝强大的体液免疫系统息息相关。
（2）栉孔扇贝血淋巴甘露聚糖结合凝集素的分离纯化。本论文依次通过硫酸铵盐析和甘露聚糖-亲和层析柱亲和层析，从栉孔扇贝血淋巴上清中分离纯化出甘露聚糖结合凝集素，通过对其凝集活性的检测以及蛋白分子量的分析发现：提纯的MBL可以凝集鳗弧菌和毕赤酵母，对高等动物血细胞以及其它检测菌体并无凝集活性；MBL在Native-PAGE中的分子量为645kDa，在SDS-PAGE中的分子量为73kDa，表明该凝集素是由一种亚基组成的多聚体。
（3）抗栉孔扇贝C-型凝集素Cflec290单克隆抗体的制备和筛选。本论文首先通过体外重组表达获得重组蛋白rCfLec290，免疫小鼠后，经细胞融合技术制备了抗rCfLec290单克隆抗体，并分别通过ELISA技术和Western blotting技术对抗体进行筛选，最后筛选出3株可稳定表达单抗的细胞株1A2、1A7和2B6。
（4）C-型凝集素Cflec290在栉孔扇贝血细胞及其它不同组织中的分布。首先通过半定量RT-PCR技术检测Cflec290mRNA在血细胞以及其它不同组织中的表达含量，再通过制备地高辛标记的Cflec290的cRNA核酸探针，检测血细胞滴片以及其它不同组织切片中mRNA表达的位置，最后通过抗重组蛋白rCfLec290单克隆抗体检测该凝集素在血细胞滴片及不同组织切片中的分布情况。结果发现：通过半定量RT-PCR，Cflec290在血细胞以及其它各组织中均有表达，但在外套膜、肾脏以及性腺中的转录水平最高，血细胞、鳃和肝胰腺次之，在肌肉中的转录水平较弱；原位杂交显示，阳性信号主要发现在栉孔扇贝的外套膜表皮细胞以及肝胰腺管壁细胞中，在其它组织中并未发现；通过利用抗重组蛋白rCfLec290的单克隆抗体的间接免疫荧光检测发现，CfLec290蛋白主要分布在扇贝血细胞膜，以及鳃、外套膜、肝胰腺等组织表皮和腔体表面。结果表明，Cflec290可在栉孔扇贝血细胞、外套膜表皮细胞和肝胰腺管壁细胞中合成与表达，功能蛋白则广泛分布于扇贝血细胞及各组织器官中，在血细胞膜，鳃、外套膜、肝胰腺表皮，腔体表面尤为丰富。依此推测，血细胞、外套膜和肝胰腺在Cflec290合成和表达过程中发挥着重要作用，该凝集素在血细胞、外套膜或肝胰腺细胞内合成后，被分泌到细胞外，伴随着体液流动，被运输到全身多组织器官中行使功能。
Lectins contribute a lot to the scallop humoral immune system for avoidingthe pathogen invasion and environmental stimulus. They play an important role in thepathogen recognition, as well as the physiological process of hemostasis, coagulation,material transport and wound healing in scallops. In this dissertation, the agglutinationactivities of the cell-free haemolymph (CFH) and haemocyte, gills, mantle,hepatopancreas, gonad, kidney, adductor muscle of C. farreri were tested separatelyagainst the rabbit blood cells (RBC), mouse blood cells (MBC) and the fluoresceinisothiocyanate-labelled microbes, and a mannan-binding lectin (MBL) was purifiedfrom CFH by affinity chromatography.; and then, monoclonal antibodies wereproduced, and they were used to analyzed the CfLec290distribution characteristics ofC. farreri haemocytes as well as other organizations combined with RT-PCR and insitu hybridization.
(1) Agglutination activities of haemolymph and tissue extracts in scallop C.farreri were tested. In this dissertation, cell-free haemolymph (CFH) and crudeextracts from the haemocytes, gills, mantle, hepatopancreas, gonad, kidney andadductor muscle of C. farreri were obtained, and their agglutination activities weretested separately against RBC, MBC and fluorescein isothiocyanate-labeled microbes.Agglutination inhibition were carried out with EDTA and saccharides. The resultsshowed the CFH could aggregate Vibrio anguillarum, Staphylococcus aureus,Bacillus subtilis, Pichia pastoris and MBC, and the agglutination could be inhibitedby DEAE dextran, EDTA, D-mannose-6-phosphate (M6P), N-acetyl glucosamine(GlcNAc), mannan, zymosan A, chondroitin sulphate, peptidoglycan (PGN) andlipopolysaccharides (LPS); Gill extracts aggregated S. aureus, B. subtilis and P.pastoris, and the agglutination activity could be inhibited by EDTA, N-acetyl galactosamine (GalNAc), mannan, zymosan A, chondroitin sulphate, PGN and LPS;Mantle, hepatopancreas, gonad and kidney extracts could only aggregate B. subtilis,and this could be inhibited by mannan, zymosan A and PGN; however, noagglutination activity was detected using haemocyte or adductor muscle extracts. Theresults indicated that multiple types of lectins exist in C. farreri, and CFH has thehighest agglutination activity.
(2) Mannan-binding lectin (MBL) was purified from haemolymph of C.farreri.MBL was purified from CFH by affinity chromatography using a mannan-sepharosecolumn and analyzed using native-and denaturing-PAGE. The results showed that thepurified MBL could aggregate Vibrio anguillarum, and exhibited a molecular mass of645kDa using native-PAGE and73kDa using SDS-PAGE, suggesting that C. farreriMBL is an polymer composed of one subunit.
(3) Production of the monoclonal antibodies against rCfLec290. Therecombinant protein rCfLec290were produced and purified in vitro. And then thepurified rCfLec290was used to immunize the mouse, and after the cell fusion, ELISAand western blotting were employed to screen the fusion cells, Finally,3hybridomaswere achieved, MAb1A2,1A7and2B6.
(4) Distribution of C-type lectin Cflec290in C. farreri were detected. First,Semi-quantitative RT-PCR and in situ hybridization was employed to detect theCflec290mRNA distribution, and the monoclonal antibodies against rCfLec290wereproduced to detect the distribution of the protein CfLec290in different tissue by IIFAand western blotting. The results showed that: In semi-quantitative RT-PCR, Cflec290mRNA expressed at the highest level in mantle, kidneys and gonads, moderate in gillsand adductor muscles, least in hepatopancreas; In ISH, positive signals were mainlyobserved in the epithelium of mantle and hepatopancreas cells; In IIFA, the positivefluorescent signals were mainly distributed in the membrane of haemocytes, and theedge of gills, mantle, kidney and glands.
引文
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