表达人CD47基因的巴马小型猪创建及其表达分析

异种器官移植是解决供体器官短缺的有效途径之一,猪(Sus scrofa)是人类(Homo sapiens)异种器官移植的理想供体.然而,异种移植物进入受体后将被受体巨噬细胞清除,其主要原因是巨噬细胞表面的受体-信号调节蛋白α(singal regulatory proteinα,SIRPα)不能特异地识别异种细胞表面的整合素相关蛋白(integrin associated protein,IAP又称CD47),导致移植物细胞被受体内的巨噬细胞吞噬并清除.本研究在敲除α-1,3-半乳糖基转移酶基因(α-1,3-galactosyltransferase knockout,GTKO)的巴马小型猪基础上,转入hCD47基因,制备表达hCD47基因的GTKO/hCD47巴马小型猪,可避免超急性排斥反应并减弱受体巨噬细胞对移植物细胞的吞噬.首先构建巨细胞病毒增强子和鸡β肌动蛋白启动子(cytomegalovirus early enhancer/chicken β-actin promoter,CAG)调控的hCD47基因表达载体pCAGGS-hCD47-Neo,转染GTKO巴马小型猪的耳成纤维细胞,然后通过体细胞克隆技术制备转基因克隆猪.利用PCR对克隆仔猪进行转基因鉴定,通过qRT-PCR、Westem blot和免疫组化等方法分析hCD47在转基因猪的心脏、肝脏、脾脏、肺脏、肾脏、胰腺等组织的表达.抽取存活仔猪血液进行生理指标检测,监测其健康状况.通过体细胞克隆技术获得了13头转基因克隆猪,PCR鉴定均为hCD47基因阳性猪.qRT-PCR结果显示,hCD47基因在各器官中的表达量由高至低依次为胰腺、肝脏、肺脏、脾脏、肾脏和心脏.Western blot及免疫组化结果进一步证实hCD47基因在胰腺、肝脏等中均具有较强表达,与qRT-PCR结果一致.血液生理指标显示,存活仔猪健康状况良好.本研究成功制备了表达hCD47基因的GTKO巴马小型猪,确证了hCD47基因在主要器官中的表达特征,将有助于探究hCD47基因在减弱受体巨噬细胞对异种移植各器官的排斥反应.     

P-selectin on activated platelets promotes hematogenous metastasis of primary lung cancer via P-selectin glycoprotein ligand-1

AIM: To investigate the relationship between platelets/P-selectin on activated platelets and clinico-pathological features, hematogenous metastasis and prognosis of lung cancer, and to explore the effect of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interaction on the hematogenous metastasis-related integrin β3 (ITGB3). METHODS: The expression of P-selectin on activated platelets was detected by flow cytometry, and its effects on lung cancer and the risk of hematogenous metastasis were analyzed. The expression of PSGL-1 in different types of lung cancer tissues and cell lines was determined by Western blot. By co-culturing platelets and lung cancer cells in vitro, the effects of up- and down-regulation of PSGL-1 on invasion and migration abilities of lung cancer cells were observed. RESULTS: The peripheral blood platelet counts and P-selectin expression on activated platelets in the patients with lung cancer were significantly increased (P<0.05). The P-selectin expression on activated platelets was significantly associated with hematogenous metastasis of lung cancer (r=0.256, P<0.05). The strongest expression of PSGL-1 was found in the lung adenocarcinoma samples, next in the lung squamous-cell carcinoma samples, and the weakest in small-cell lung cancer samples. P-selectin promoted transmembrane invasion of lung cancer cells. Inhibition of P-selectin and its ligand PSGL-1 reduced ITGB3 expression, invasion and migration of lung cancer cells. CONCLUSION: The P-selectin level on activated platelets is significantly associated with hematogenous metastasis of lung cancer, which is related to the binding of P-selectin and its ligand PSGL-1. Up-regulation of ITGB3 level after their binding might be one of the mechanisms of the remodeling of extracellular matrix to facilitate hematogenous metastasis of lung cancer.     

口蹄疫病毒受体羊源整联蛋白β6亚基配体结合域的原核表达、纯化及鉴定

利用原核细胞表达羊口蹄疫病毒(FMDV)受体整联蛋白β6亚基的配体结合域 (LBD)片段,分离纯化目的蛋白.以含有羊整联蛋白β6亚基全长cDNA的质粒为模板,PCR扩增得到β6LBD基因片段,经酶切消化处理后与同样酶切处理的原核表达载体pP_(RO)EX~(TM)HTb相连,构建原核表达质粒pP_(RO)/β6LBD,测序确认读码框正确后,将其转化感受态细胞 BL21 (DE3),IPTG诱导表达重组羊β6LBD融合蛋白.SDS-PAGE鉴定重组蛋白的表达并利用镍离子亲和树脂对其纯化,通过酶联免疫吸附试验(ELISA)和Western blot方法分析鉴定表达产物.成功构建了pPRO/β6LBD原核表达载体,实现了羊FMDV受体整联蛋白亚基β6LBD在大肠杆菌中的高效表达, SDS-PAGE显示其相对分子质量(Mr)约为33 kDa,重组蛋白主要以包涵体形式存在于菌体中.用本试验室保存的抗FMDV受体猪源整联蛋白β6亚基LBD的单克隆抗体进行ELISA 和Western blot检测,显示该单抗可与重组目的蛋白发生特异性反应,证明纯化后的蛋白与特异性抗体具有良好的特异性反应及抗原活性.为深入研究整联蛋白受体β6亚基在羊体内的分布及其在FMDV致病过程中的作用机制奠定了基础.     

牛整合素β5亚基基因的构建与原核表达

[目的]构建牛整合素β5亚基（ITGB5）基因,并进行原核表达。[方法]扩增目的基因,将其重组到pET-32a（＋）质粒中,将经鉴定为阳性的重组克隆,转化大肠杆菌感受态细胞表达蛋白后纯化。[结果]从牛甲状腺、扁桃体扩增到目的基因ITGB5;ITGB5基因表达产物的SDS-PAGE结果显示,在88 KD处出现表达产物及纯化后的蛋白;W estern-blot分析表明,抗血清可与原核表达的蛋白特异性结合。[结论]表达、并纯化了ITGB5蛋白。     

大竹蛏β-整合素基因SgβInt的特性分析和重组表达

整合素作为一种细胞粘附因子,在细胞粘附、迁移、增殖、凋亡和吞噬等过程中发挥重要作用。本研究利用分子克隆技术获得了大竹蛏(Solen grandis)β-整合素基因(SgβInt)的c DNA全长,分析了其编码氨基酸序列的进化特点,并以SWISS-MODEL预测了氨基酸序列的三级结构。SgβInt基因序列全长为1168 bp,5'和3'非编码区(UTR)分别为61 bp和18 bp,开放阅读框(ORF)为1089 bp,编码362个氨基酸,理论等电点约为4.98,分子量为30.0 kDa。通过荧光定量PCR法检测了SgβInt在健康大竹蛏各组织中以及大竹蛏受到脂多糖、肽聚糖和葡聚糖等微生物多糖刺激后的表达。结果显示,SgβInt在血细胞、鳃、肝胰腺、性腺、肌肉和外套膜等组织中均可表达,在鳃中的表达量最高;脂多糖、肽聚糖和葡聚糖都可以诱导SgβInt表达量上调,SgβInt的表达量分别在脂多糖和葡聚糖刺激后的3 h和48 h达到最高;肽聚糖刺激后SgβInt表达量上调幅度最大,在6 h时达到最高,证实SgβInt参与大竹蛏抵御外源微生物的免疫应答。利用基因重组技术构建了SgβInt的重组表达载体,为进一步分析软体动物整合素的功能、揭示大竹蛏先天性免疫机制奠定了基础。