华支睾吸虫sPLA2相互作用蛋白质的筛选鉴定及其功能研究

发布时间：2018-05-23 08:45

  本文选题：华支睾吸虫 + 分泌型磷脂酶A2　； 参考：《中山大学》2016年博士论文

【摘要】：华支睾吸虫(Clonorchis sinensis,Cs)是一种致病性吸虫,主要由食入感染华支睾吸虫囊蚴的“鱼生”感染,成虫寄生于人及其他哺乳动物肝胆管系统,虫体的机械性刺激和分泌/排泄物可以刺激胆管上皮及结缔组织增生,引起胆道炎症、胆管阻塞、甚至胆管癌、肝硬化、肝癌,全世界均有患者发病和死亡,但主要流行于东亚地区包括中国、韩国和越南。目前中国有1300万患者左右。近年来,华支睾吸虫病发病呈上升趋势,已成为我国流行最广、危害最大的食源性寄生虫病之一,2005年广东省已将其列为重点防治的三大地方病之一,2006年卫生部已将其列为重点防治的寄生虫病。对华支睾吸虫引起肝纤维化和肝硬化的分子机制进行深入研究,对防治华支睾吸虫病具有重要的意义。肝纤维化是由于肝细胞外基质(ECM)合成和降解失衡,ECM过度沉积而发生的,肝星状细胞(HSC)活化、增殖是肝纤维化的关键事件。已有实验证明华支睾吸虫分泌排泄产物(Cs ESP)通过使肝星状细胞活化增殖导致肝纤维化产生,还可使胆管上皮细胞(BEC)增生、癌变致胆管癌的发生,其中Cs ESP的成分之一分泌型磷脂酶A2(s PLA2)能引起HSC的活化增殖,这种作用不能被其酶活性的作用底物卵磷脂所阻断,而只能被其特异性抗体所阻断,显示Css PLA2发挥使HSC活化增殖的作用不是通过酶活性而是通过结合相关受体实现的,但与其作用的受体蛋白及其活化HSC的具体分子机制仍不清楚。因此探究华支睾吸虫分泌型磷脂酶A2(Css PLA2)相互作用的蛋白质及其相关的信号通路,对进一步了解华支睾吸虫引起肝纤维化的有重要意义。研究目的和意义基于探究和华支睾吸虫分泌型磷脂酶A2(Css PLA2)相互作用的蛋白质及与其相互作用相关的信号通路,对进一步了解该蛋白分子在华支睾吸虫引起肝纤维化中的作用有重要意义。本课题以下列研究为目标,初步探讨Css PLA2相互作用蛋白质的筛选与其功能:(1)酵母双杂交初步筛选出与Css PLA2相互作用蛋白质;(2)对筛选出与Css PLA2相互作用蛋白质进行免疫共沉淀、GST Pull-down等体外实验进一步验证;(3)探究Css PLA2及其相互作用蛋白质结合后对人肝星状细胞系LX2细胞效应及其可能涉及的相关细胞信号通路。研究方法1.利用酵母双杂交筛选与Css PLA2相互作用的蛋白质。构建诱饵载体(p GBKT7-s PLA2),并转化到酵母菌Y2HGold,诱饵蛋白自身激活测试、毒性测试及表达验证后,含诱饵载体的酵母Y2HGold与含人肝c DNA文库载体(p GADT7)的酵母Y187进行杂交(mating),杂交后的产物涂布于营养缺陷型培养基生长,选取生长出的酵母菌落,扩大培养提取质粒后PCR产物送测序。对测序结果进行初步分析,筛选出和Css PLA2直接相互作用蛋白质。2.免疫共沉淀(CO-IP)验证Css PLA2及筛选出的蛋白质间相互作用将Css PLA2及相互作用候选蛋白质TM7SF3分别克隆到真核表达载体p EGFP和pc DNA6,构建好的重组载体利用Lipofectamine 2000瞬时共转染转染到293T细胞,以单独转染重组Css PLA2表达载体和重组相互作用蛋白质表达载体作为对照组。转染成功后293T细胞以RIPA液裂解,细胞裂解液与Protein A琼脂糖珠在4°C共孵育过夜后离心沉淀后洗涤,再用抗c-Myc抗体及抗PLA2抗体分别进行Western blotting分析。3.GST Pull-down验证Css PLA2及筛选出的蛋白质间相互作用将Css PLA2及相互作用蛋白质TM7SF3分别克隆到表达载体p MAL-c2x和p GEX-4T-1,在大肠杆菌中表达,用亲和层析法纯化目的蛋白,并做SDS-PAGE分析。带MBP标签的Css PLA2和带GST标签的蛋白质GST-TM7SF3混合物用Amylose resin进行亲和层析纯化,以MBP蛋白、GST蛋白分别与MBP-Css PLA2混合后在Amylose resin中亲和层析纯化的产物作为对照组,纯化后的产物均用抗GST抗体进行Western blotting分析。4.对Css PLA2相互作用蛋白质在LX2细胞上进行免疫组化定位免疫组化法测定Css PLA2相互作用蛋白质TM7SF3在LX2细胞上的细胞定位,LX2细胞与或不与抗TM7SF3抗体孵育后经苏木素染色,洗涤后观察细胞染色情况及黄褐色颗粒分布位置。5.检测Css PLA2及筛选出的蛋白质对LX2细胞的作用及其涉及的相关信号通路LX2细胞分别用重组Css PLA2蛋白、重组Css PLA2及抗TM7SF3抗体混合物及PBS孵育24h后,CCK-8法分别测定LX2细胞增殖情况,实时荧光定量PCR检测TGF-β、ERK、JNK and NF-κB等与肝纤维化相关的信号通路的表达变化。研究结果1.酵母双杂交筛选与Cs PLA2相互作用的蛋白质酵母双杂交初步筛选出5个可能与Css PLA2相互作用的蛋白质,其中1个定位于细胞膜(TM7SF3)、1个为分泌蛋白质(hemopexin,isoform CRA_a)、3个定位于线粒体(metallothionein-2、aminoacylase-1 isoform b、cytochrome c oxidase subunit I)。排除定位于线粒体不能与Css PLA2直接结合的蛋白质以及分泌游离于血液中的蛋白质,选定有可能与Css PLA2直接作用的定位于细胞膜上的蛋白TM7SF3进行进一步验证。2.CO-IP验证Css PLA2及筛选出的蛋白质间相互作用重组后Css PLA2和TM7SF3蛋白均能在293T细胞中表达。单独转染p EGFP-Css PLA2的293T细胞裂解液经Protein A agarose进行免疫沉淀再洗涤后只能被抗PLA2抗体识别,单独转染pc DNA6-TM7SF3细胞裂解液经Protein A agarose进行免疫沉淀再洗涤后既不能被抗c-Myc抗体识别,也不能被抗PLA2抗体识别,只有p EGFP-Css PLA2和pc DNA6-TM7SF3共转染后的293T细胞裂解液经Protein A agarose进行免疫沉淀再洗涤后,能被抗c-Myc抗体和抗PLA2抗体同时识别,显示Css PLA2和TM7SF3蛋白存在相互作用。3.GST Pull-down验证Css PLA2及筛选出的蛋白质间相互作用MBP-Css PLA2和GST-TM7SF3在大肠杆菌中表达,相对分子质量分别约为42k Da和70k Da。只有MBP-Css PLA2和GST-TM7SF3蛋白质混合物经只能沉积含MBP标签蛋白Amylose resinpull down后的产物能被抗GST抗体识别,Css PLA2和TM7SF3蛋白存在相互作用。4.对Css PLA2相互作用蛋白质在LX2细胞上进行免疫组化定位免疫组化法测定Css PLA2相互作用蛋白质TM7SF3在LX2细胞上的细胞定位,LX2细胞与或不与抗TM7SF3抗体孵育后经苏木素染色,洗涤后观察细胞染色情况及黄褐色颗粒分布位置。只有与抗TM7SF3抗体孵育后LX2细胞显现棕黄色,且黄褐色颗粒主要集中于细胞膜上。5.检测Css PLA2及筛选出的蛋白质对LX2细胞的增殖作用及有关的信号通路CCK-8检测结果显示Css PLA2孵育24h后相比PBS对照组OD450结果显示PBS孵育组LX2细胞OD450约为1.2,Css PLA2孵育组LX2细胞OD450约为1.5,LX2细胞增殖率较PBS组升高了25%,而Css PLA2和抗TM7SF3抗体混合物孵育组LX2细胞OD450约为1.0,细胞增殖率较Css PLA2孵育组降低了33%。结果显示Css PLA2能够使LX2细胞增殖,抗体阻断TM7SF3后Css PLA2对LX2细胞增殖作用减弱,间接验证了TM7SF3和Css PLA2共同作用使LX2细胞增殖。抗TM7SF3抗体阻断TM7SF3后,Css PLA2对LX2细胞活化增殖作用受抑制。Css PLA2孵育LX2细胞24h后,相比空白对照组,肝纤维化相关信号通路,包括ERK、JNK、NF-k B、TGF-Smad等通路关键基因表达水平也上调,抗TM7SF3抗体阻断TM7SF3后,相比Css PLA2孵育LX2细胞上述通路关键基因表达水平下调。结论1.酵母双杂交筛选出和Css PLA2相互作用的人蛋白质TM7SF3等。2.免疫共沉淀、GSTPull-down进一步验证Css PLA2与人TM7SF3的相互作用。3.免疫组化对TM7SF3在人肝星状细胞系LX2细胞上定位,主要位于细胞膜。4.通过TM7SF3抗体阻断等反向验证Css PLA2致肝纤维化所涉及的相关信号通路,包括ERK、JNK、NF-k B、TGF-Smad等通路。
[Abstract]:Clonorchis sinensis (Cs) is a kind of pathogenic fluke, which mainly infects the "fish students" infected with the cysts of Clonorchis sinensis, the adult parasites in human and other mammalian hepatobiliary system. The mechanical stimulation and secretion / excretion of the body can stimulate the hyperplasia of bile duct epithelium and connective tissue, cause biliary inflammation and bile duct. Obstruction, even cholangiocarcinoma, liver cirrhosis, liver cancer, and death in all the world, but mainly in East Asia, including China, Korea and Vietnam. At present, there are 13 million patients in China. In recent years, the incidence of Clonorchis sinensis has been on the rise, which has become one of the most widespread and hardest food borne parasites in China in 2005. Guangdong province has been listed as one of the three major endemic diseases in key prevention and control. In 2006, the Ministry of health has listed it as a major parasitic disease. The molecular mechanism of liver fibrosis and cirrhosis caused by Clonorchis sinensis is very important for the prevention and control of Clonorchis sinensis. The liver fibrosis is due to the liver extracellular matrix (ECM). It has been proved that the secretion and excretory product of Clonorchis sinensis (Cs ESP) is produced by the activation and proliferation of hepatic stellate cells (Cs ESP), and the proliferation of hepatic stellate cells (Cs ESP), and the proliferation of bile duct epithelial cells (BEC) and carcinogenesis of cholangiocarcinoma. One of the components of Cs ESP, secretory phospholipase A2 (s PLA2), can cause the activation and proliferation of HSC, which can not be blocked by its enzyme activity as a substrate for phosphatidylcholine, but can only be blocked by its specific antibody. It shows that Css PLA2 plays the role of activating HSC not by enzyme activity but by binding to related receptors. However, the specific molecular mechanism of its receptor protein and its activation of HSC is still unclear. Therefore, exploring the protein and related signaling pathway of the interaction of the secretory phospholipase A2 (Css PLA2) of Clonorchis sinensis is of great significance to the further study of the liver fibrosis caused by Clonorchis sinensis. The purpose and significance of this study are based on the exploration and China. The proteins interacting with the secretory phospholipase A2 (Css PLA2) of Clonorchis Clonorchis and the signaling pathway related to their interaction are of great significance for further understanding the role of this protein molecule in the liver fibrosis caused by Clonorchis sinensis. The following research aims at the preliminary study of the screening of Css PLA2 interaction protein and its work. (1) a preliminary screening of the interaction protein with Css PLA2 by yeast two hybrid; (2) further verification of the immune coprecipitation and GST Pull-down screening with Css PLA2 interaction proteins; (3) exploring the LX2 cell effect of Css PLA2 and its interacting protein binding to the human hepatic stellate cell line and its possible phase Cell signaling pathway. Study method 1. use yeast two hybrid to screen proteins interacting with Css PLA2. Construct bait carrier (P GBKT7-s PLA2), and convert to yeast Y2HGold, bait protein self activation test, toxicity test and expression validation, yeast Y2HGold containing bait carrier and human liver C DNA library carrier (P GADT7). Yeast Y187 was used to hybridize (mating). The products after hybridization were coated on the growth of nutrient deficient culture medium. The growing yeast colony was selected and the PCR products were sequenced. The sequencing results were preliminarily analyzed, and the direct interaction of Css PLA2 with.2. immunoprecipitation (CO-IP) was selected to verify Css PLA2 and to be screened out. Css PLA2 and the interaction candidate protein TM7SF3 were cloned to the eukaryotic expression vector p EGFP and PC DNA6, respectively. The recombinant vector was transfected to 293T cells by instantaneous co transfection by Lipofectamine 2000, and the recombinant Css PLA2 expression vector and recombinant protein expression vector were transfected as a pair. After the transfection, the 293T cells were lysed with RIPA solution, the cell lysate and Protein A agarose beads were incubated at 4 degree C for the night after the centrifuge precipitation. The Western blotting analysis of the Western blotting analysis.3.GST Pull-down was used to verify the.3.GST Pull-down and the interaction between the proteins and the screened proteins. Protein TM7SF3 was cloned to express vector p MAL-c2x and P GEX-4T-1, expressed in Escherichia coli, purified the target protein by affinity chromatography, and made SDS-PAGE analysis. Css PLA2 with MBP label and GST-TM7SF3 mixture with GST label were purified by affinity chromatography with Amylose. The products purified by affinity chromatography in Amylose resin were used as the control group, and the purified products were all Western blotting analysis with anti GST antibody and.4. on Css PLA2 interacting protein on LX2 cells, immunohistochemical localization method was used to determine the cell determination of Css PLA2. After incubation with or without anti TM7SF3 antibody, LX2 cells were stained with hematoxylin, and after washing, the staining of cells and the distribution of yellow brown particles were observed by.5.. The effects of Css PLA2 and the selected proteins on LX2 cells and related signaling pathways were used for LX2 cells, recombinant Css PLA2 protein, Css PLA2 and anti TM7SF3 antibodies. After the mixture and PBS were incubated for 24h, the proliferation of LX2 cells was measured by CCK-8 method, and the expression of TGF- beta, ERK, JNK and NF- kappa B and so on were detected by real-time fluorescent quantitative PCR. The results of 1. yeast two hybrid screening for protein yeast two hybrid screening with the interaction of Cs 1 of the interacting proteins are located in the cell membrane (TM7SF3), the 1 are secreting proteins (hemopexin, isoform CRA_a), and the 3 are located in the mitochondria (metallothionein-2, aminoacylase-1 isoform B, cytochrome c oxidase subunit). Protein in liquid, the protein TM7SF3, which is located directly on the membrane of Css PLA2, is selected for further verification by.2.CO-IP verification that the interaction of Css PLA2 and selected proteins can be expressed in 293T cells after the recombination of Css PLA2 and TM7SF3 protein. After immunoprecipitation and re washing, A agarose can only be identified by anti PLA2 antibody. PC DNA6-TM7SF3 cell lysate can not be identified by anti c-Myc antibody and can not be identified by anti PLA2 antibody after Protein A agarose. After immunoprecipitation and re washing, Protein A agarose can be identified by anti c-Myc and anti PLA2 antibodies. The interaction of Css PLA2 and TM7SF3 protein exists in the presence of.3.GST Pull-down to verify that Css PLA2 and selected proteins are interacted and expressed in Escherichia coli. And 70K Da. only MBP-Css PLA2 and GST-TM7SF3 protein mixture can be identified by anti GST antibody after only Amylose resinpull down containing MBP label protein, Css PLA2 and the interaction of the proteins exist. The interaction protein TM7SF3 was located on the cells of LX2 cells. LX2 cells were stained with or not incubated with anti TM7SF3 antibodies by hematoxylin. After washing, the staining of cells and the distribution position of the yellow brown granules were observed. Only after incubating with anti TM7SF3 antibody, the LX2 cells appeared brown yellow, and the yellow brown granules were mainly concentrated on the cell membrane.5. to detect Css. The effect of PLA2 and selected protein on the proliferation of LX2 cells and the related signal pathway CCK-8 detection results showed that Css PLA2 incubated 24h compared to the PBS control group OD450 results showed that LX2 cell OD450 in the PBS incubating group was about 1.2, and that the proliferation rate of Css cells was about 25%. The LX2 cell OD450 in the antibody mixture incubated group was about 1, and the cell proliferation rate was lower than that of Css PLA2 incubating group. The 33%. results showed that Css PLA2 could increase the proliferation of LX2 cells. After the antibody blocked TM7SF3, Css PLA2 could weaken the proliferation of LX2 cells. The effect of PLA2 on LX2 cell activation and proliferation was inhibited by.Css PLA2 incubating LX2 cells 24h. Compared with the blank control group, the key gene expression levels of liver fibrosis related signaling pathway, including ERK, JNK, NF-k B, TGF-Smad and so on, were also up-regulated. Conclusion 1. yeast two hybrids are used to screen the.2. immunoprecipitation of human protein TM7SF3, which interact with Css PLA2, and GSTPull-down further verifies the interaction of Css PLA2 and human TM7SF3 by.3. immunohistochemistry on TM7SF3 in the LX2 cells of human hepatic stellate cell line, which is mainly located in the reverse validation of cell membrane.4. through the blocking of antibodies. LA2 related signaling pathways involved in liver fibrosis include ERK, JNK, NF-k B, TGF-Smad and other pathways.
【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R383.2
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本文编号：1924054